Treatment of inflammatory diseases using placental stem cells

ABSTRACT

Provided herein are methods of treatment of individuals having an immune-related disease, disorder or condition, for example, inflammatory bowel disease, graft-versus-host disease, multiple sclerosis, rheumatoid arthritis, psoriasis, lupus erythematosus, diabetes, mycosis fungoides (Alibert-Bazin syndrome), or scleroderma using placental stem cells or umbilical cord stem cells.

This application claims benefit of U.S. Provisional Application No.60/901,067, filed Feb. 12, 2007, which is incorporated herein in itsentirety by reference.

1. FIELD

Provided herein are methods of using human placental stem cells to treatindividuals having a disease, disorder or condition caused by, orrelating to, an unwanted or harmful immune response, for example,inflammatory bowel disease, graft-versus-host disease, multiplesclerosis, rheumatoid arthritis, psoriasis, lupus erythematosus,diabetes, mycosis fungoides (Alibert-Bazin syndrome), or scleroderma.

2. BACKGROUND

Human stem cells are totipotential or pluripotential precursor cellscapable of generating a variety of mature human cell lineages. Evidenceexists that demonstrates that stem cells can be employed to repopulatemany, if not all, tissues and restore physiologic and anatomicfunctionality.

Many different types of mammalian stem cells have been characterized.See, e.g., Caplan et al., U.S. Pat. No. 5,486,359 (human mesenchymalstem cells); Boyse et al., U.S. Pat. No. 5,004,681 (fetal and neonatalhematopoietic stem and progenitor cells); Boyse et al., U.S. Pat. No.5,192,553 (same); Beltrami el al., Cell 114(6):763-766 (2003) (cardiacstem cells); Forbes et al., J. Pathol. 197(4):510-518 (2002) (hepaticstem cells). Umbilical cord blood, and total nucleated cells derivedfrom cord blood, have been used in transplants to restore, partially orfully, hematopoietic function in patients who have undergone ablativetherapy.

The placenta is a particularly attractive source of stem cells. Becausemammalian placentas are plentiful and are normally discarded as medicalwaste, they represent a unique source of medically-useful stem cells.Provided herein are such isolated placental stem cells, populations ofthe placental stem cells, and methods of using the same to treatdisease, disorder or condition caused by, or relating to, an unwanted ordeleterious immune response, for example, inflammatory bowel disease,graft-versus-host disease, multiple sclerosis, rheumatoid arthritis,psoriasis, lupus erythematosus, diabetes, mycosis fungoides(Alibert-Bazin syndrome), or scleroderma.

3. SUMMARY

Provided herein are methods of treating, managing, ameliorating orpreventing diseases, disorders and/or conditions associated with orcaused by an immune response, e.g., associated with, resulting in orcaused by inflammation. In one embodiment, provided herein is a methodof treating an individual having or at risk of developing a disease,disorder or condition associated with or caused by harmful, deleterious,inappropriate or unwanted immune response, e.g., inflammation,comprising administering to the individual a therapeutically effectiveamount of placental stem cells, or medium conditioned by placental stemcells, wherein the therapeutically effective amount is an amountsufficient to cause a detectable improvement in one or more symptoms of,or a reduction in the progression of one or more symptoms of, saiddisease, disorder or condition. Also provided herein is the use ofplacental stem cells in the manufacture of a medicament for treating,managing, ameliorating or preventing diseases, disorders and/orconditions associated with or caused by an immune response, e.g.,associated with, resulting in or caused by inflammation. In a specificembodiment, said placental stem cells are CD10⁺, CD34⁻, CD105⁺, CD200⁺placental stem cells. In another specific embodiment, said placentalstem cells express CD200 and HLA-G, or express CD73, CD105, and CD200,or express CD200 and OCT-4, or express CD73, CD105 and HLA-G, or expressCD73 and CD105 and facilitate the formation of one or more embryoid-likebodies in a population of placental cells comprising said stem cell whensaid population is cultured under conditions that allow for theformation of an embryoid-like body, or express OCT-4 and (c) facilitatethe formation of one or more embryoid-like bodies in a population ofplacental cells comprising said stem cell when said population iscultured under conditions that allow for the formation of anembryoid-like body. In a more specific embodiment, the placental stemcells suppress the activity of an immune cell, e.g., suppressproliferation of a T cell.

In a specific embodiment, said disease, disorder or condition is aninflammatory bowel disease. In a more specific embodiment, saidinflammatory bowel disease is Crohn's disease. In another more specificembodiment, said Crohn's disease is gastroduodenal Crohn's disease. Inanother more specific embodiment, said Crohn's disease is jejunoileitis.In another more specific embodiment, said Crohn's disease is ileitis. Inanother more specific embodiment, said Crohn's disease is ileocolitis.In another more specific embodiment, said Crohn's disease is Crohn'scolitis. In another specific embodiment, said inflammatory bowel diseaseis ulcerative colitis.

In a more specific embodiment, said symptom of inflammatory boweldisease is one or more of inflammation and swelling of a part of the GItract, abdominal pain, frequent emptying of the bowel, diarrhea, rectalbleeding, anemia, weight loss, arthritis, skin problems, fever,thickening of the intestinal wall, formation of scar tissue in theintestines, formation of sores or ulcers in the intestine, developmentof one or more fistulas in the intestinal wall, development of one ormore fissures in the anus, development of a nutritional deficiency,development of kidney stones, development of gallstones, development ofa disease of the liver or biliary system, bloody diarrhea, nausea,abdominal cramps, anemia, fatigue, weight loss, loss of appetite, lossof bodily fluids and nutrients, skin lesions, joint pain, growthfailure, development of osteoporosis, or eye inflammation.

In another specific embodiment, said symptom of inflammatory boweldisease, and is one or more of pruritic or painful rash, fever,generalized erythroderma, desquamation, raised (e.g., higher thannormal) levels of bilirubin, raised levels of alanine aminotransferase(ALT), raised levels of aspartate aminotransferase (AST), raised levelsof alkaline phosphatase (AP), diarrhea, internal bleeding, cramping,abdominal pain, and ileus, burning sensation in the eye, eye irritation,photophobia, eye pain due to decreased tear secretion; dryness of themouth, sensitivity to spicy or acidic foods, abdominal pain, dysphagia,odynophagia, weight loss, obstructive lung disease, muscular weakness,neuropathic pain, or muscle cramps.

In another specific embodiment, said disease, disorder or condition isgraft-versus-host disease. In a more specific embodiment, saidgraft-versus-host disease develops after an allogeneic bone marrowtransplant. In another more specific embodiment, said graft-versus-hostdisease develops after a solid organ transplant. In another morespecific embodiment, said graft-versus-host disease develops after acomposite tissue allograft. In another more specific embodiment, saidgraft-versus-host disease is reduced in grade by at least one step bysaid administering. In another more specific embodiment, saidgraft-versus-host disease does not progress beyond grade II within 100days after transplantation as a result of said administering. In anothermore specific embodiment, said graft-versus-host disease does notprogress beyond grade I within 100 days after transplantation as aresult of said administering.

In another specific embodiment, said disease, disorder or condition isrheumatoid arthritis (RA). In a more specific embodiment, theadministration is sufficient to cause a detectable improvement in one ormore symptoms of RA, or sufficient to detectably reduce the onset of oneor more symptoms of RA, in at least one joint in the individual with RA.In another specific embodiment, the administration is sufficient tocause a detectable improvement in one or more symptoms of RA, orsufficient to detectably reduce the onset of one or more symptoms of RA,in at least one non-joint tissue in the individual with RA. In a morespecific embodiment, said non-joint tissue is skin (dermis), lungs,autoimmune system or blood, renal tissue, cardiovascular tissue, oculartissue, or neurological tissue. In a more specific embodiment, saidsymptom of RA is a condition adjunct to RA. In a more specificembodiment, said condition adjunct to RA is pyoderma gangrenosum,neutrophilic dermatosis, Sweet's syndrome, viral infection, erythemanodosum, lobular panniculitis, atrophy of digital skin, palmar erythema,diffuse thinning (rice paper skin), skin fragility, subcutaneous noduleson an exterior surface, e.g., on the elbows, fibrosis of the lungs(e.g., as a consequence of methotrexate therapy), Caplan's nodules,vasculitic disorders, nail fold infarcts, neuropathy, nephropathy,amyloidosis, muscular pseudohypertrophy, endoscarditis, left ventricularfailure, valulitis, scleromalacia, mononeuritis multiplex, atlanto-axialsubluxation. In another specific embodiment of the method, a pluralityof the placental or umbilical cord stem cells has been geneticallyengineered to express a fusion protein comprising IL-1Ra and DHFR.

In another specific embodiment, the disease, disorder or condition ismultiple sclerosis. In a more specific embodiment, said multiplesclerosis is relapsing/remitting multiple sclerosis, secondaryprogressive multiple sclerosis, primary progressive multiple sclerosis,or progressive/relapsing multiple sclerosis. In another specificembodiment, said symptom of multiple sclerosis is one or more of asensory disturbance in a limb, optic nerve dysfunction, pyramidal tractdysfunction, bladder dysfunction, bowel dysfunction, sexual dysfunction,ataxia, or diplopia.

In another specific embodiment, the disease, disorder or condition islupus erythematosus. In a more specific embodiment, said symptom oflupus erythematosus is one or more of malar rash, butterfly rash,discoid lupus, alopecia, mouth, nasal, and vaginal ulcers, lesions onthe skin, joint pain anemia and/or iron deficiency, lower than normalplatelet and white blood cell counts, antiphospholipid antibodysyndrome, presence of anticardiolipin antibody in the blood,pericarditis, myocarditis, endocarditis, lung and/or pleuralinflammation, pleuritis, pleural effusion, lupus pneumonitis, chronicdiffuse interstitial lung disease, pulmonary hypertension, pulmonaryemboli, pulmonary hemorrhage, painless hematuria or proteinuria, lupusnephritis, renal failure, and/or development of membranousglomerulonephritis with “wire loop” abnormalities); neurologicalmanifestations (e.g., seizures, psychosis, abnormalities in thecerebrospinal fluid); T-cell abnormalities (e.g., deficiency in CD45phosphatase and/or increased expression of CD40 ligand); and/ornonspecific manifestations (e.g., lupus gastroenteritis, lupuspancreatitis, lupus cystitis, autoimmune inner ear disease,parasympathetic dysfunction, retinal vasculitis, systemic vasculitis,increased expression of FcεRIγ, increased and sustained calcium levelsin T cells, increase of inositol triphosphate in the blood, reduction inprotein kinase C phosphorylation, reduction in Ras-MAP kinase signaling,and/or a deficiency in protein kinase A I activity.

In another specific embodiment, said disease, disorder or condition isscleroderma. In a more specific embodiment, the scleroderma is diffusescleroderma. In a more specific embodiment, the scleroderma is limitedscleroderma (CREST syndrome). In a more specific embodiment, thescleroderma is morphea/linear scleroderma. In another more specificembodiment, said symptom is one or more of hardening of the skin of theface, hardening of the skin of the fingers, Reynaud's syndrome,inappropriate vasoconstriction in an extremity, calcinosis,telangiectasia, esophageal dysmotility, or presence in the blood of ananti-centromere antibody or an anti-scl70/anti-topoisomerase antibody.In another more specific embodiment, the method comprises administeringa second therapeutic agent to said individual. In a more specificembodiment, said second therapeutic agent is an anti-inflammatory drug,a proton pump inhibitor, an immunosuppressant compound, or avasodilator.

In another specific embodiment, said disease, disorder or condition ismycosis fungoides (Alibert-Bazin syndrome). In a more specificembodiment, said mycosis fungoides is in the patch phase. In a morespecific embodiment, said mycosis fungoides is in the skin tumor phase.In another more specific embodiment, said mycosis fungoides is in theskin redness (erythroderma) stage. In another more specific embodiment,said mycosis fungoides is in the lymph node stage. In another morespecific embodiment, said symptom is one or more of development of flat,red patches that are itchy; development of flat, red patches that areraised and hard (plaques); development of raised lumps (nodules) appear;development of large red, itchy, scaly areas over the body; cracking ofthe skin of the palms and soles; thickening of the skin of the palms andsoles; and crack; or inflammation of the lymph nodes. In another morespecific embodiment, the method comprises administering a secondtherapeutic agent to said individual. In a more specific embodiment,said second therapeutic agent is an anti-inflammatory drug, animmunosuppressant compound, exposure to sunlight, exposure toultraviolet light, a topical steroid, local superficial radiotherapy,total skin electron beam radiation, application of organic honey to skinaffected by erythroderma, an interferon, a retinoid, a rexinoid, orvorinostat.

In another embodiment, said disease, disorder or condition is diabetes.In a specific embodiment, said diabetes is diabetes mellitus Type 1.

In another embodiment, said disease, disorder or condition is psoriasis.In a more specific embodiment, the psoriasis is plaque psoriasis(psoriasis vulgaris). In another more specific embodiment, saidpsoriasis is flexural psoriasis (inverse psoriasis). In another morespecific embodiment, said psoriasis is guttate psoriasis. In anothermore specific embodiment, said psoriasis is pustular psoriasis. Inanother more specific embodiment, said psoriasis is nail psoriasis. Inanother more specific embodiment, said psoriasis is psoriatic arthritis.In another more specific embodiment, said psoriasis is erythrodermicpsoriasis. In another specific embodiment, the therapeutically effectiveamount of placental stem cells or umbilical cord stem cells, or culturemedium conditioned by placental stem cells or umbilical cord stem cellsis an amount sufficient to cause a 5, 10, 15, 20, 25, 30, 35, 40 or morepoint reduction in the Psoriasis Area Severity Index. In one embodiment,the invention provides for the administration of an effective doseplacental stem cells to an individual affected with psoriasis, whereinsaid effective dose is an amount of placental stem cells sufficient,e.g., to cause a detectable improvement in, reduce the severity of, orreduce the progression of, one or more of the symptoms of psoriasis. Ina more specific embodiment, said one or more symptoms is development ofone or more of raised areas of inflamed skin covered with silvery whitescaly skin; development of plaques; smooth inflamed patches of skinoccurring in skin folds; development of one or more small oval spots;development of one or more pustules; a change in the appearance of oneor more finger nails or toe nails; onycholysis; crumbling of one or morenails; joint and connective tissue inflammation; dactylitis;spondylitis; widespread inflammation and exfoliation of the skin overmost of the body surface; or severe itching, swelling and/or pain. Inanother specific embodiment, the method comprises additionallyadministering one or more therapeutic agents or therapies, wherein saidtherapeutic agents or therapies comprise one or more of a cream orointment comprising a corticosteroid, a cream or ointment comprising avitamin D₃ analog, a cream or ointment comprising a anthralin, a creamor ointment comprising a argan oil, a cream or ointment comprising aretinoid, or a cream or ointment comprising coal tar; one or moreexposures, e.g., for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18or 20 minutes, to ultraviolet light, e.g., UVB of a wavelength betweenabout 280 nm to about 315 nm, particularly about 311 nm to about 312 nm;topical administration of psoralen in combination with exposure to UVAlight; or one or more systemic administrations of one or more ofmethotrexate, cyclosporine, a retinoid, tioguanine, hydroxyurea,sulfasalazine, mycophenolate mofetil, azathioprine, oral tacrolimusand/or a fumaric acid ester.

In another specific embodiment of any of the above methods, the methodcomprises administration of a second therapeutic agent to the individualhaving the disease, disorder or condition. In a more specificembodiment, said second therapeutic agent is an anti-inflammatory agent,an immunomodulatory agent, and immunosuppressive agent, a painmedication, or an antibiotic. In a more specific embodiment, the secondtherapeutic agent is an immunomodulatory agent. In a more specificembodiment, said immunomodulatory agent is an immune suppressant. In aneven more specific embodiment, said immune suppressive agent is ananti-CD3 antibody (e.g., OKT3, muronomab), an anti-IL-2 receptorantibody (e.g., basiliximab (SIMTJILECT®) and daclizumab (ZENAPAX®)), ananti T cell receptor antibody (e.g., Muromonab-CD3), azathioprine, acalcineurin inhibitor, a corticosteroid, cyclosporine, methotrexate,mercaptopurine, mycophenolate mofetil, tacrolimus, or sirolimus. Inanother more specific embodiment, the second therapeutic agent comprisesa stem cell of another type, e.g., a bone marrow-derived mesenchymalstem cell, bone marrow, or a hematopoietic stem cell.

3.1 Definitions

As used herein, the term “SH2” refers to an antibody that binds anepitope on the marker CD105. Thus, cells that are referred to as SH2⁺are CD105⁺.

As used herein, the terms “SH3” and SH4″ refer to antibodies that bindepitopes present on the marker CD73. Thus, cells that are referred to asSH3⁺ and/or SH4⁺ are CD73⁺.

As used herein, the term “isolated stem cell” means a stem cell that issubstantially separated from other, non-stem cells of the tissue, e.g.,placenta, from which the stem cell is derived. A stem cell is “isolated”if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of thenon-stem cells with which the stem cell is naturally associated areremoved from the stem cell, e.g., during collection and/or culture ofthe stem cell.

As used herein, the term “isolated population of cells” means apopulation of cells that is substantially separated from other cells ofthe tissue, e.g., placenta, from which the population of cells isderived. A population of, e.g., stem cells is “isolated” if at least50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the cells with whichthe population of stem cells are naturally associated are removed fromthe population of stem cells, e.g., during collection and/or culture ofthe population of stem cells.

As used herein, the term “placental stem cell” refers to a stem cell orprogenitor cell that is derived from a mammalian placenta, regardless ofmorphology, cell surface markers, or the number of passages after aprimary culture, which adheres to a tissue culture substrate (e.g.,tissue culture plastic or a fibronectin-coated tissue culture plate).The term “placenta stem cell” as used herein does not, however, refer toa trophoblast, a cytotrophoblast, embryonic germ call, or embryonic stemcell, as those cells are understood by persons of skill in the art. Acell is considered a “stem cell” if the cell retains at least oneattribute of a stem cell, e.g., a marker or gene expression profileassociated with one or more types of stem cells; the ability toreplicate at least 10-40 times in culture; multipotency, e.g., theability to differentiate, either in vitro, in vivo or both, into cellsof one or more of the three germ layers; the lack of adult (i.e.,differentiated) cell characteristics, or the like. The terms “placentalstem cell” and “placenta-derived stem cell” may be used interchangeably.Unless otherwise noted herein, the term “placental” includes theumbilical cord. The placental stem cells disclosed herein are, incertain embodiments, multipotent in vitro (that is, the cellsdifferentiate in vitro under differentiating conditions), multipotent invivo (that is, the cells differentiate in vivo), or both.

As used herein, a stem cell is “positive” for a particular marker whenthat marker is detectable. For example, a placental stem cell ispositive for, e.g., CD73 because CD73 is detectable on placental stemcells in an amount detectably greater than background (in comparison to,e.g., an isotype control). A cell is also positive for a marker whenthat marker can be used to distinguish the cell from at least one othercell type, or can be used to select or isolate the cell when present orexpressed by the cell.

As used herein, “immunomodulation” and “immunomodulatory” mean causing,or having the capacity to cause, a detectable change in an immuneresponse, and the ability to cause a detectable change in an immuneresponse.

As used herein, “immunosuppression” and “immunosuppressive” meancausing, or having the capacity to cause, a detectable reduction in animmune response, and the ability to cause a detectable suppression of animmune response.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 : Viability of placental stem cells from perfusion (A), amnion(B), chorion (C), or amnion-chorion plate (D), or umbilical cord stemcells (E). Numbers on X-axis designate placenta from which stem cellswere obtained.

FIG. 2 : Percent HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells from perfusion (A),amnion (B), chorion (C), or amnion-chorion plate (D), or umbilical cordstem cells (E) as determined by FACSCalibur. Numbers on X-axis designateplacenta from which stem cells were obtained.

FIG. 3 : Percent HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells from perfusion (A),amnion (B), chorion (C), or amnion-chorion plate (D), or umbilical cordstem cells (E), as determined by FACS Aria. Numbers on X-axis designateplacenta from which stem cells were obtained.

FIG. 4 : HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from placental perfusate.

FIG. 5 : HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from amnion.

FIG. 6 : HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from chorion.

FIG. 7 : HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from amnion-chorion plate.

FIG. 8 : HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from umbilical cord.

FIG. 9 : Average expression of HLA-G, CD10, CD13, CD33, CD38, CD44,CD90, CD105, CD117, CD200 expression in stem cells derived fromperfusion (A), amnion (B), chorion (C), amnion-chorion plate (D) orumbilical cord (E).

FIGS. 10A and 10B: The mixed lymphocyte reaction (MLR) is a model forthe naïve immune response, and is inhibited by placental stem cells.From the gated “live” and CD8⁺ and CD4⁺ T cell gates, the percentage ofcarboxyfluorescein succinimidyl ester (CFSE)^(LOW) cells was monitored(FIGS. 10A and 10B, respectively).

FIG. 11 : Placenta-derived stem cells from amnion chorionic plate (AC)and umbilical cord stroma (UC) suppress the allo-MLR. The MLR isperformed with either CD4⁺ T cells, CD8⁺ T cells, or equal amounts ofCD4⁺ and CD8⁺ T cells. Abscissa: percent suppression of proliferation.

FIG. 12A and FIG. 12B: Placental stem cells and umbilical cord stemcells inhibit the allo-MLR. A six day assay in round bottom 96 wellplate wells. Placental cells:T cells:Dendritic cells=approximately1:10:1. Stem cells were obtained from amnion-chorion (AC), amnioticmembrane (AM) or umbilical cord (UC). FB=fibroblast. BM=bonemarrow-derived mesenchymal stem cells.

FIG. 13 : Placental stem cells from different donors suppress theallo-MLR to different extents. The figure compares suppression in an MLRby placental stem cells from two placental donors, designated 61665 and63450. Stem cells from placenta 63450 appears to suppress the MLR to agreater degree than stem cells from placenta 61665.

FIG. 14 : Seventeen day regression assay and the modified placental stemcell regression assay. The x axis represents the number of placentalstem cells added to the assay. The number of surviving CD23⁺ LCL(lymphoblastoid cell line, an artificially-created transformed B cellline) is measured on the Y axis.

FIG. 15 : Placental stem cell suppression of T cell proliferation in thesix day regression assay. A regression assay was set up using CFSEstained T cells. After six days, T cell proliferation was assessed.Relative suppression of T cell proliferation by stem cells fromamnion-chorion (AC), umbilical cord (UC), amniotic membrane (AM), orbone marrow (BM) is shown.

FIG. 16 : Percentage change in suppression on introduction of transwellinsert in the MLR, separating placental cells from T cells but allowingexchange of culture medium. Umbilical cord stem cells at 25,000, 50,000,75,000 or 100,000 per reaction show both a relatively high degree ofsuppression and a relatively high degree of need for cell to cellcontact in the high titers to accomplish the suppression.

FIG. 17 : Umbilical cord stroma stem cells (UC) added at 12,500 (UCOP/TW 12.5) to 100,000 (UC OP/TW 100) were either separated from the NLRby a membrane (TW) or in contact with the MLR (OP). Equal numbers ofCD4⁺ T cells and CD8⁺ T cells were used, and the percentage suppressionof the MLR (% CFSE^(Low)=89%) was calculated.

FIG. 18 : The relationship between placental stem cell dose and cell tocell contact dependency is not linear. The changes in MLR suppression onintroduction of the insert are calculated from the values given in FIG.17 .

FIG. 19 : Differential suppression of T cell responses by placental stemcells and BMSCs. The degree of suppression conferred by placental stemcells or BMSCs was calculated comparing the percentage of MLR T cells inthe CFSE^(Lo) gate, more than 70%, to that of the adherent cell MLRs.The MLR was either separated from the adherent cells (transwell), or wasperformed in an open well (open). The X axis gives the numbers ofadherent cells, in thousands, added to 500,000 T cells and 50,000 DCs.The ratio of adherent cells to T cells goes from 1:5 to 1:40.

FIG. 20 : Differential cell to cell contact requirements for placentalstem cell and bone marrow-derived stem cell immune suppression. From thesuppression data given in FIG. 15 , the contact dependency wascalculated and displayed against the adherent cell/T cell ratio (n=3,except UC: n=2).

FIG. 21 : T regulatory cells are not required for placental stemcell-mediated T cell suppression. A regression assay was performed usingeither whole PBMCs or PBMCs depleted of T regulatory cells, both CFSEstained, adding UC placental stem cells to some conditions. N=1.

FIG. 22 : CFSE^(Hi) cells proliferate in a secondary MLR. From anplacental stem cell MLR using CFSE stained cells, the CFSE^(Hi) T cellswere isolated on a FACS Aria. The cells were used in an MLR. N=1.

FIG. 23 : Supernatant from suppressed stem cell MLR does not suppressMLR at 75% replacement. UC (PUC), AC (PAC), and BMSC (PBM) MLRs wereperformed, and all suppressed the MLR more than 50%. Supernatant fromthe experiments were used to replace from 10 to 150 μl of the 200 μlmedium used for a fresh MLR. As controls, medium from T cell and AC(T/AC) or T cell and bone marrow-derived stem cell (T/BM) cocultureswere also used in the same way (N=2).

FIGS. 24A, 24B: Pre-incubating T cells and adherent cells does notinfluence MLR suppression. T cells from 2 donors were used in twoindependent experiments. Mature DCs (A) or CFSE stained CD3⁺ T cells (B)were incubated with umbilical cord stem cells (UC) or bonemarrow-derived stem cells for the indicated number of days before addingDCs (on day 0, A) or CFSE CD3⁺ T cells (B, thereby starting the MLR. Theadherent cell MLRs then proceeded for six days, as normal. N=2.

FIGS. 25A, 25B: A. MIP-1α and MIP-1β secretion in the MLR, and MLR withplacental stem cells or bone marrow-derived stem cells, correlatesinversely with MLR suppression. B: T cell and NK cell CFSE data from thesame experiment. Supernatants were harvested from the MLR shown in FIG.24B, and analyzed for MIP-1α and MIP-1β: MLR was performed as described,and on average 55% (T cells) or 83% (NK cells) CFSE^(Low) cells wereobserved. The suppressive effect of stem cell addition was calculated.N=2 (NK part: N=1).

FIG. 26 : In the modified regression assay and MLR supernatants, MCP-1was measured. The placental stem cell suppression of the MLR andregression assay correlates with secretion of the chemoattractant MCP-1.AC: stem cells from amnion-chorion plate. UC: stem cells from umbilicalcord. Light bars: MLR assay results. Dark bars: regression assayresults. Y axis: pg of MCP-1 in assay solution.

FIG. 27 : IL-6 measurement in the supernatant of the modified MLR andregression assay. The placental stem cell suppression of the MLR andregression assay correlates with IL-6 secretion. AC: stem cells fromamnion-chorion plate. UC: stem cells from umbilical cord. Light bars:MLR assay results. Dark bars: regression assay results. Y axis: pg ofIL-6 in assay solution.

5. DETAILED DESCRIPTION

Provided herein are methods for the treatment of an individual having adisease, disorder or condition associated with, arising from, or relatedto an inappropriate, unwanted, harmful or deleterious immune response,e.g., an autoimmune disease, comprising administering to the individualhaving the disease, disorder or condition one or more doses of placentalstem cells and/or umbilical cord stem cells. Methods for the treatmentof such individuals, and for the administration of such stem cells,alone or in combination with other therapies, are discussed in detailbelow.

5.1 Immunomodulation Using Placental Stem Cells

Provided herein are methods for the modulation, e.g., suppression, ofthe activity, e.g., proliferation, of an immune cell, or plurality ofimmune cells, by contacting the immune cell(s) with a plurality ofplacental stem cells. Such immunomodulation is useful in the treatmentof an individual having a disease, disorder or condition caused by, orrelating to, an unwanted or harmful immune response, for example,inflammatory bowel disease, graft-versus-host disease, multiplesclerosis, rheumatoid arthritis, psoriasis, lupus erythematosus,diabetes, mycosis fungoides (Alibert-Bazin syndrome), or scleroderma.Such immunomodulation is also useful, e.g., in the reduction orelimination of a host immune response against allogenic tissue, e.g., atransplanted organ, composite tissue allograft, and the like.

In one embodiment, provided herein is a method of suppressing an immuneresponse comprising contacting a plurality of immune cells with aplurality of placental stem cells for a time sufficient for saidplacental stem cells to detectably suppress an immune response, whereinsaid placental stem cells detectably suppress T cell proliferation in amixed lymphocyte reaction (MLR) assay or a regression assay.

Placental stem cells are, e.g., the placental stem cells describedelsewhere herein (see Section 5.2). Placental stem cells used forimmunosuppression can be derived or obtained from a single placenta ormultiple placentas. Placental stem cells used for immunosuppression canalso be derived from a single species, e.g., the species of the intendedrecipient or the species of the immune cells the function of which is tobe reduced or suppressed, or can be derived from multiple species.

An “immune cell” in the context of this method means any cell of theimmune system, particularly T cells and NK (natural killer) cells. Thus,in various embodiments of the method, placental stem cells are contactedwith a plurality of immune cells, wherein the plurality of immune cellsare, or comprises, a plurality of T cells (e.g., a plurality of CD3⁺ Tcells, CD4⁺ T cells and/or CD8⁺ T cells) and/or natural killer cells. An“immune response” in the context of the method can be any response by animmune cell to a stimulus normally perceived by an immune cell, e.g., aresponse to the presence of an antigen. In various embodiments, animmune response can be the proliferation of T cells (e.g., CD3⁺ T cells,CD4⁺ T cells and/or CD8⁺ T cells) in response to a foreign antigen, suchas an antigen present in a transfusion or graft, or to a self-antigen,as in an autoimmune disease. The immune response can also be aproliferation of T cells contained within a graft. The immune responsecan also be any activity of a natural killer (NK) cell, the maturationof a dendritic cell, or the like. The immune response can also be alocal, tissue- or organ-specific, or systemic effect of an activity ofone or more classes of immune cells, e.g., the immune response can begraft versus host disease, inflammation, formation ofinflammation-related scar tissue, an autoimmune condition (e.g.,rheumatoid arthritis, Type I diabetes, lupus erythematosus, etc.). andthe like.

“Contacting” in this context encompasses bringing the placental stemcells and immune cells together in a single container (e.g., culturedish, flask, vial, etc.) or in vivo, for example, the same individual(e.g., mammal, for example, human). In a preferred embodiment, thecontacting is for a time sufficient, and with a sufficient number ofplacental stem cells and immune cells, that a change in an immunefunction of the immune cells is detectable. More preferably, in variousembodiments, said contacting is sufficient to suppress immune function(e.g., T cell proliferation in response to an antigen) by at least 50%,60%, 70%, 80%, 90% or 95%, compared to the immune function in theabsence of the placental stem cells. Such suppression in an in vivocontext can be determined in an in vitro assay (see below); that is, thedegree of suppression in the in vitro assay can be extrapolated, for aparticular number of placental stem cells and a number of immune cellsin a recipient individual, to a degree of suppression in the individual.

In certain embodiments, provided herein are methods of using placentalstem cells to modulate an immune response, or the activity of aplurality of one or more types of immune cells, in vitro. Contacting theplacental stem cells and plurality of immune cells can comprisecombining the placental stem cells and immune cells in the same physicalspace such that at least a portion of the plurality of placental stemcells interacts with at least a portion of the plurality of immunecells; maintaining the placental stem cells and immune cells in separatephysical spaces with common medium; or can comprise contacting mediumfrom one or a culture of placental stem cells or immune cells with theother type of cell (for example, obtaining culture medium from a cultureof placental stem cells and resuspending isolated immune cells in themedium). In a specific example, the contacting is performed in a MixedLymphocyte Reaction (MLR). In another specific example, the contactingis performed in a regression assay.

Such contacting can, for example, take place in an experimental settingdesigned to determine the extent to which a particular plurality ofplacental stem cells is immunomodulatory, e.g., immunosuppressive. Suchan experimental setting can be, for example, a mixed lymphocyte reaction(MLR) or regression assay. Procedures for performing the MLR andregression assays are well-known in the art. See, e.g. Schwarz, “TheMixed Lymphocyte Reaction: An In Vitro Test for Tolerance,” J. Exp. Med.127(5):879-890 (1968); Lacerda et al., “Human Epstein-Barr Virus(EBV)-Specific Cytotoxic T Lymphocytes Home Preferentially to and InduceSelective Regressions of Autologous EBV-Induced B Lymphoproliferationsin Xenografted C.B-17 Scid/Scid Mice,” J. Exp. Med. 183:1215-1228(1996). In a preferred embodiment, an MLR is performed in which aplurality of placental stem cells are contacted with a plurality ofimmune cells (e.g., lymphocytes, for example, CD3⁺, CD4⁺ and/or CD8⁺ Tlymphocytes).

The MLR can be used to determine the immunosuppressive capacity of aplurality of placental stem cells. For example, a plurality of placentalstem cells can be tested in an MLR comprising combining CD4⁺ or CD8⁺ Tcells, dendritic cells (DC) and placental stem cells in a ratio of about10:1:2, wherein the T cells are stained with a dye such as, e.g., CFSEthat partitions into daughter cells, and wherein the T cells are allowedto proliferate for about 6 days. The plurality of placental stem cellsis immunosuppressive if the T cell proliferation at 6 days in thepresence of placental stem cells is detectably reduced compared to Tcell proliferation in the presence of DC and absence of placental stemcells. In such an MLR, placental stem cells are either thawed orharvested from culture. About 20,000 placental stem cells areresuspended in 100 μl of medium (RPMI 1640, 1 mM HEPES buffer,antibiotics, and 5% pooled human serum), and allowed to attach to thebottom of a well for 2 hours. CD4⁺ and/or CD8⁺ T cells are isolated fromwhole peripheral blood mononuclear cells Miltenyi magnetic beads. Thecells are CFSE stained, and a total of 100,000 T cells (CD4⁺ T cellsalone, CD8⁺ T cells alone, or equal amounts of CD4⁺ and CD8⁺ T cells)are added per well. The volume in the well is brought to 200 μl, and theMLR is allowed to proceed.

In one embodiment, therefore, provided herein is a method of suppressingan immune response comprising contacting a plurality of immune cellswith a plurality of placental stem cells for a time sufficient for saidplacental stem cells to detectably suppress T cell proliferation in amixed lymphocyte reaction (MLR) assay or in a regression assay. In oneembodiment, said placental stem cells used in the MLR represent a sampleor aliquot of placental stem cells from a larger population of placentalstem cells.

Populations of placental stem cells obtained from different placentas,or different tissues within the same placenta, can differ in theirability to modulate an activity of an immune cell, e.g., can differ intheir ability to suppress T cell activity or proliferation or NK cellactivity. It is thus desirable to determine, prior to use, the capacityof a particular population of placental stem cells forimmunosuppression. Such a capacity can be determined, for example, bytesting a sample of the placental stem cell population in an MLR orregression assay. In one embodiment, an MLR is performed with thesample, and a degree of immunosuppression in the assay attributable tothe placental stem cells is determined. This degree of immunosuppressioncan then be attributed to the placental stem cell population that wassampled. Thus, the MLR can be used as a method of determining theabsolute and relative ability of a particular population of placentalstem cells to suppress immune function. The parameters of the MLR can bevaried to provide more data or to best determine the capacity of asample of placental stem cells to immunosuppress. For example, becauseimmunosuppression by placental stem cells appears to increase roughly inproportion to the number of placental stem cells present in the assay,the MLR can be performed with, in one embodiment, two or more numbers ofplacental stem cells, e.g., 1×10³, 3×10³, 1×10⁴ and/or 3×10⁴ placentalstem cells per reaction. The number of placental stem cells relative tothe number of T cells in the assay can also be varied. For example,placental stem cells and T cells in the assay can be present in anyratio of, e.g. about 10:1 to about 1:10, preferably about 1:5, though arelatively greater number of placental stem cells or T cells can beused.

The regression assay can be used in similar fashion.

Provided herein are methods of using placental stem cells to modulate animmune response, or the activity of a plurality of one or more types ofimmune cells, in vivo. Placental stem cells and immune cells can becontacted, e.g., in an individual that is a recipient of a plurality ofplacental stem cells. Where the contacting is performed in anindividual, in one embodiment, the contacting is between exogenousplacental stem cells (that is, placental stem cells not derived from theindividual) and a plurality of immune cells endogenous to theindividual. In specific embodiments, the immune cells within theindividual are CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, and/or NKcells.

Such immunosuppression using placental stem cells would be advantageousfor any condition caused or worsened by, or related to, an inappropriateor undesirable immune response. Placental stem cell-mediatedimmunomodulation, e.g., immunosuppression, would, for example, be usefulin the suppression of an inappropriate immune response raised by theindividual's immune system against one or more of its own tissues. Invarious embodiments, therefore, provided herein is a method ofsuppressing an immune response, wherein the immune response is anautoimmune disease, e.g., lupus erythematosus, diabetes, rheumatoidarthritis, or multiple sclerosis.

The contacting of the plurality of placental stem cells with theplurality of one or more types of immune cells can occur in vivo in thecontext of, or as an adjunct to, for example, grafting or transplantingof one or more types of tissues to a recipient individual. Such tissuesmay be, for example, bone marrow or blood; an organ; a specific tissue(e.g., skin graft); composite tissue allograft (i.e., a graft comprisingtwo or more different types of tissues); etc. In this regard, theplacental stem cells can be used to suppress one or more immuneresponses of one or more immune cells contained within the recipientindividual, within the transplanted tissue or graft, or both. Thecontacting can occur before, during and/or after the grafting ortransplanting. For example, placental stem cells can be administered atthe time of the transplant or graft. The placental stem cells can also,or alternatively, be administered prior to the transplanting orgrafting, e.g., about 1, 2, 3, 4, 5, 6 or 7 days prior to thetransplanting or grafting. Placental stem cells can also, oralternatively, be administered to a transplant or graft recipient afterthe transplantation or grafting, for example, about 1, 2, 3, 4, 5, 6 or7 days after the transplanting or grafting. Preferably, the plurality ofplacental stem cells are contacted with the plurality of placental stemcells before any detectable sign or symptom of an immune response,either by the recipient individual or the transplanted tissue or graft,e.g., a detectable sign or symptom of graft-versus-host disease ordetectable inflammation, is detectable.

In another embodiment, the contacting within an individual is primarilybetween exogenous placental stem cells and exogenous progenitor cells orstem cells, e.g., exogenous progenitor cells or stem cells thatdifferentiate into immune cells. For example, individuals undergoingpartial or full immunoablation or myeloablation as an adjunct to cancertherapy can receive placental stem cells in combination with one or moreother types of stem or progenitor cells. For example, the placental stemcells can be combined with a plurality of CD34⁺ cells, e.g., CD34⁺hematopoietic stem cells. Such CD34⁺ cells can be, e.g., CD34⁺ cellsfrom a tissue source such as peripheral blood, umbilical cord blood,placental blood, or bone marrow. The CD34⁺ cells can be isolated fromsuch tissue sources, or the whole tissue source (e.g., units ofumbilical cord blood or bone marrow) or a partially purified preparationfrom the tissue source (e.g., white blood cells from cord blood) can becombined with the placental stem cells. Combinations of placental stemcells and cord blood, or stem cells from cord blood, are described inHariri, U.S. Application Publication No. 2003/0180269.

The placental stem cells can be administered to the individual in aratio, with respect to the known or expected number of immune cells,e.g., T cells, in the individual, of from about 10:1 to about 1:10,preferably about 1:5. However, a plurality of placental stem cells canbe administered to an individual in a ratio of, in non-limitingexamples, about 10,000:1, about 1,000:1, about 100:1, about 10:1, about1:1, about 1:10, about 1:100, about 1:1,000 or about 1:10,000.Generally, about 1×10⁵ to about 1×10⁸ placental stem cells per recipientkilogram, preferably about 1×10⁶ to about 1×10⁷ placental stem perrecipient kilogram can be administered to effect immunosuppression. Invarious embodiments, a plurality of placental stem cells administered toan individual or subject comprises at least, about, or no more than,1×10⁵, 3×10⁵, 1×10⁶, 3×10⁶, 1×10⁷, 3×10⁷, 1×10⁸, 3×10⁸, 1×10⁹, 3×10⁹placental stem cells, or more.

The placental stem cells can also be administered with one or moresecond types of stem cells, e.g., mesenchymal stem cells from bonemarrow. Such second stem cells can be administered to an individual withplacental stem cells in a ratio of, e.g., about 1:10 to about 10:1.

To facilitate contacting the placental stem cells and immune cells invivo, the placental stem cells can be administered to the individual byany route sufficient to bring the placental stem cells and immune cellsinto contact with each other. For example, the placental stem cells canbe administered to the individual, e.g., intravenously, intramuscularly,intraperitoneally, intraocularly, parenterally, or directly into anorgan, e.g., pancreas. For in vivo administration, the placental stemcells can be formulated as a pharmaceutical composition, as described inSection 5.6.1, below.

The method of immunosuppression can additionally comprise the additionof one or more immunosuppressive agents, particularly in the in vivocontext. In one embodiment, the plurality of placental stem cells arecontacted with the plurality of immune cells in vivo in an individual,and a composition comprising an immunosuppressive agent is administeredto the individual. Immunosuppressive agents are well-known in the artand include, e.g., anti-T cell receptor antibodies (monoclonal orpolyclonal, or antibody fragments or derivatives thereof), anti-IL-2receptor antibodies (e.g., Basiliximab (SIMULECT®) or daclizumab(ZENAPAX)®), anti T cell receptor antibodies (e.g., Muromonab-CD3),azathioprine, corticosteroids, cyclosporine, tacrolimus, mycophenolatemofetil, sirolimus, calcineurin inhibitors, and the like. In a specificembodiment, the immunosuppressive agent is a neutralizing antibody tomacrophage inflammatory protein (MIP)-1α or MIP-1β. Preferably, theanti-MIP-1α or MIP-1β antibody is administered in an amount sufficientto cause a detectable reduction in the amount of MIP-1α and/or MIP-1β insaid individual, e.g., at the time of transplanting.

5.2 Placental Stem Cells and Placental Stem Cell Populations

The methods of immunosuppression provided herein use placental stemcells, that is, stem cells obtainable from a placenta or part thereof,that (1) adhere to a tissue culture substrate; (2) have the capacity todifferentiate into non-placental cell types; and (3) have, in sufficientnumbers, the capacity to detectably suppress an immune function, e.g.,proliferation of CD4⁺ and/or CD8⁺ T cells in a mixed lymphocyte reactionassay or regression assay. Placental stem cells are not derived fromblood, e.g., placental blood or umbilical cord blood. The placental stemcells used in the methods and compositions provided herein have thecapacity, and are selected for their capacity, to suppress the immunesystem of an individual.

Placental stem cells can be either fetal or maternal in origin (that is,can have the genotype of either the mother or fetus). Populations ofplacental stem cells, or populations of cells comprising placental stemcells, can comprise placental stem cells that are solely fetal ormaternal in origin, or can comprise a mixed population of placental stemcells of both fetal and maternal origin. The placental stem cells, andpopulations of cells comprising the placental stem cells, can beidentified and selected by the morphological, marker, and culturecharacteristics discussed below.

5.2.1 Physical and Morphological Characteristics

The placental stem cells used as described herein, when cultured inprimary cultures or in cell culture, adhere to the tissue culturesubstrate, e.g., tissue culture container surface (e.g., tissue cultureplastic). Placental stem cells in culture assume a generallyfibroblastoid, stellate appearance, with a number of cytoplasmicprocesses extending from the central cell body. The placental stem cellsare, however, morphologically differentiable from fibroblasts culturedunder the same conditions, as the placental stem cells exhibit a greaternumber of such processes than do fibroblasts. Morphologically, placentalstem cells are also differentiable from hematopoietic stem cells, whichgenerally assume a more rounded, or cobblestone, morphology in culture.

5.2.2 Cell Surface, Molecular and Genetic Markers

Placental stem cells, and populations of placental stem cells, useful inthe methods and compositions provided herein, express a plurality ofmarkers that can be used to identify and/or isolate the stem cells, orpopulations of cells that comprise the stem cells. The placental stemcells, and stem cell populations (that is, two or more placental stemcells) include stem cells and stem cell-containing cell populationsobtained directly from the placenta, or any part thereof (e.g., amnion,chorion, placental cotyledons, and the like). Placental stem cellpopulations also includes populations of (that is, two or more)placental stem cells in culture, and a population in a container, e.g.,a bag. Placental stem cells are not, however, trophoblasts.

Placental stem cells generally express the markers CD73, CD105, CD200,HLA-G, and/or OCT-4, and do not express CD34, CD38, or CD45. Placentalstem cells can also express HLA-ABC (MHC-1) and HLA-DR. These markerscan be used to identify placental stem cells, and to distinguishplacental stem cells from other stem cell types. Because the placentalstem cells can express CD73 and CD105, they can have mesenchymal stemcell-like characteristics. However, because the placental stem cells canexpress CD200 and HLA-G, a fetal-specific marker, they can bedistinguished from mesenchymal stem cells, e.g., bone marrow-derivedmesenchymal stem cells, which express neither CD200 nor HLA-G. In thesame manner, the lack of expression of CD34, CD38 and/or CD45 identifiesthe placental stem cells as non-hematopoietic stem cells.

In one embodiment, provided herein is an isolated cell populationcomprising a plurality of immunosuppressive placental stem cells thatare CD200⁺, HLA-G⁺, wherein said plurality detectably suppresses T cellproliferation in a mixed lymphocyte reaction (MLR) assay. In a specificembodiment of the isolated populations, said stem cells are also CD73⁺and CD105⁺. In another specific embodiment, said stem cells are alsoCD34⁻, CD38⁻ or CD45⁻. In a more specific embodiment, said stem cellsare also CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In another embodiment,said isolated population produces one or more embryoid-like bodies whencultured under conditions that allow the formation of embryoid-likebodies.

In another embodiment, provided herein is an isolated cell populationcomprising a plurality of immunosuppressive placental stem cells thatare CD73⁺, CD105⁺, CD200⁺, wherein said plurality detectably suppress Tcell proliferation in a mixed lymphocyte reaction (MLR) assay. In aspecific embodiment of said populations, said stem cells are HLA-G⁺. Inanother specific embodiment, said stem cells are CD34⁻, CD38⁻ or CD45⁻.In another specific embodiment, said stem cells are CD34⁻, CD38⁻ andCD45⁻. In a more specific embodiment, said stem cells are CD34⁻, CD38⁻,CD45⁻, and HLA-G⁺. In another specific embodiment, said population ofcells produces one or more embryoid-like bodies when cultured underconditions that allow the formation of embryoid-like bodies.

An isolated cell population useful in the compositions and methodsprovided herein can comprise a plurality of immunosuppressive placentalstem cells that are CD200⁺, OCT-4⁺, wherein said plurality detectablysuppresses T cell proliferation in a mixed lymphocyte reaction (MLR)assay. In a specific embodiment, said stem cells are CD73⁺ and CD105⁺.In another specific embodiment, said stem cells are HLA-G⁺. In anotherspecific embodiment, said stem cells are CD34⁻, CD38⁻ and CD45⁻. In amore specific embodiment, said stem cells are CD34⁻, CD38⁻, CD45⁻,CD73⁺, CD105⁺ and HLA-G⁺. In another specific embodiment, the populationproduces one or more embryoid-like bodies when cultured under conditionsthat allow the formation of embryoid-like bodies.

Also provided herein is an isolated cell population comprising aplurality of immunosuppressive placental stem cells that are CD73⁺,CD105⁺ and HLA-G⁺, wherein said plurality detectably suppresses T cellproliferation in a mixed lymphocyte reaction (MLR) assay. In a specificembodiment of the above plurality, said stem cells are also CD34⁻, CD38⁻or CD45⁻. In another specific embodiment, said stem cells are alsoCD34⁻, CD38⁻ and CD45⁻. In another specific embodiment, said stem cellsare also OCT-4⁺. In another specific embodiment, said stem cells arealso CD200⁺ In a more specific embodiment, said stem cells are alsoCD34⁻, CD38⁻, CD45⁻, OCT-4⁺ and CD200⁺.

Further provided herein is an isolated cell population comprising aplurality of immunosuppressive placental stem cells that are CD73⁺,CD105⁺ stem cells, wherein said plurality forms one or moreembryoid-like bodies under conditions that allow formation ofembryoid-like bodies, and wherein said plurality detectably suppresses Tcell proliferation in a mixed lymphocyte reaction (MLR) assay. In aspecific embodiment, said stem cells are also CD34⁻, CD38⁻ or CD45⁻. Inanother specific embodiment, said stem cells are also CD34⁻, CD38⁻ andCD45⁻. In another specific embodiment, said stem cells are also OCT-4⁺.In a more specific embodiment, said stem cells are also OCT-4⁺, CD34⁻,CD38⁻ and CD45⁻.

Also provided herein is an isolated cell population comprising aplurality of immunosuppressive placental stem cells that are OCT-4⁺ stemcells, wherein said population forms one or more embryoid-like bodieswhen cultured under conditions that allow the formation of embryoid-likebodies, and wherein said plurality detectably suppresses T cellproliferation in a mixed lymphocyte reaction (MLR) assay. In variousembodiments, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50% at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95% of said isolated placental cells are OCT4⁺ stem cells. In aspecific embodiment of the above populations, said stem cells are CD73⁺and CD105⁺. In another specific embodiment, said stem cells are CD34⁻,CD38⁻, or CD45⁻ In another specific embodiment, said stem cells areCD200⁺. In a more specific embodiment, said stem cells are CD73⁺,CD105⁺, CD200⁺, CD34⁻, CD38⁻, and CD45⁻. In another specific embodiment,said population has been expanded, for example, passaged at least once,at least three times, at least five times, at least 10 times, at least15 times, or at least 20 times.

The immunosuppressive placental stem cells provided herein includeisolated placental stem cells, populations of isolated placental stemcells, or isolated populations of cells comprising placental stem cells,wherein the placental stem cells are CD10⁺, CD34⁻, CD105⁺ and CD200⁺.

Provided herein also is an isolated cell population comprising aplurality of immunosuppressive placental stem cells that are CD29⁺,CD44⁺, CD73⁺, CD90⁺, CD105⁺, CD200⁺, CD34⁻ and CD133⁻.

In another embodiment, stem cells useful in the compositions and methodsprovided herein are isolated placental stem cell that is HLA-A,B,C⁻,CD45⁻, CD133⁻ and CD34⁻. Further provided herein is an isolatedpopulation of placental stem cells, wherein at least about 70%, at leastabout 80%, at least about 90%, at least about 95% or at least about 99%of said placental stem cells are HLA-A,B,C⁻, CD45⁻, CD133⁻ and CD34⁻. Ina specific embodiment, said stem cell or population of placental stemcells is isolated away from placental cells that are not stem cells. Inanother specific embodiment, said population of placental stem cells isisolated away from placental stem cells that do not display thesecharacteristics. In another specific embodiment, said isolated placentalstem cell is non-maternal in origin. In another specific embodiment, atleast about 90%, at least about 95%, or at least about 99% of said cellsin said isolated population of placental stem cells, are non-maternal inorigin. In another embodiment, provided herein is a method of obtaininga placental stem cell that is HLA-A,B,C⁻, CD45⁻, CD133⁻ and CD34⁻comprising isolating said cell from placental perfusate.

In another embodiment, provided herein is an isolated placental stemcell that is CD10⁺, CD13⁺, CD33⁺, CD45⁻, CD117⁻ and CD133⁻. Furtherprovided herein is an isolated population of placental stem cells,wherein at least about 70%, at least about 80%, at least about 90%, atleast about 95% or at least about 99% of said placental stem cells areCD10⁺, CD13⁺, CD33⁺, CD45⁻, CD117⁻ and CD133⁻. In a specific embodiment,said stem cell or population of placental stem cells is isolated awayfrom placental cells that are not stem cells. In another specificembodiment, said isolated placental stem cell is non-maternal in origin.In another specific embodiment, at least about 90%, at least about 95%,or at least about 99% of said cells in said isolated population ofplacental stem cells, are non-maternal in origin. In another specificembodiment, said stem cell or population of placental stem cells isisolated away from placental stem cells that do not display thesecharacteristics. In another embodiment, provided herein is a method ofobtaining a placental stem cell that is CD10⁺, CD13⁺, CD33⁺, CD45⁻,CD117⁻ and CD133⁻ comprising isolating said cell from placentalperfusate.

In another embodiment, provided herein is an isolated placental stemcell that is CD10⁻, CD33⁻, CD44⁺, CD45⁻, and CD117⁻. Also providedherein is an isolated population of placental stem cells, wherein atleast about 70%, at least about 80%, at least about 90%, at least about95% or at least about 99% of said placental stem cells are CD10⁻, CD33⁻,CD44⁺, CD45⁻, and CD117⁻. In a specific embodiment, said stem cell orpopulation of placental stem cells is isolated away from placental cellsthat are not stem cells. In another specific embodiment, said isolatedplacental stem cell is non-maternal in origin. In another specificembodiment, at least about 90%, at least about 95%, or at least 99% ofsaid cells in said isolated population of placental stem cells, arenon-maternal in origin. In another specific embodiment, said stem cellor population of placental stem cells is isolated away from placentalstem cells that do not display these characteristics. In anotherembodiment, provided herein is a method of obtaining a placental stemcell that is CD10⁻, CD33⁻, CD44⁺, CD45⁻, CD117⁻ comprising isolatingsaid cell from placental perfusate.

In another embodiment, provided herein is an isolated placental stemcell that is CD10⁻, CD13⁻, CD33⁻, CD45⁻, and CD117⁻. Further provided isan isolated population of placental stem cells, wherein at least about70%, at least about 80%, at least about 90%, at least about 95% or atleast about 99% of said placental stem cells are CD10⁻, CD13⁻, CD33⁻,CD45⁻, and CD117⁻. In a specific embodiment, said stem cell orpopulation of placental stem cells is isolated away from placental cellsthat are not stem cells. In another specific embodiment, said isolatedplacental stem cell is non-maternal in origin. In another specificembodiment, at least about 90%, at least about 95%, or at least 99% ofsaid cells in said isolated population of placental stem cells, arenon-maternal in origin. In another specific embodiment, said stem cellor population of placental stem cells is isolated away from placentalstem cells that do not display these characteristics. In anotherembodiment, also provided herein is a method of obtaining a placentalstem cell that is CD10⁻, CD13⁻, CD33⁻, CD45⁻, and CD117⁻ comprisingisolating said cell from placental perfusate.

In another embodiment, provided herein is an isolated placental stemcell that is HLA A,B,C⁻, CD45⁻, CD34⁻, CD133⁻, positive for CD10, CD13,CD38, CD44, CD90, CD105, CD200 and/or HLA-G, and/or negative for CD117.Also provided herein is an isolated population of placental stem cells,wherein said stem cells are HLA A,B,C⁻, CD45⁻, CD34⁻, CD133⁻, and atleast about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98% or about 99% of the stem cells in the populationare positive for CD10, CD13, CD38, CD44, CD90, CD105, CD200 and/orHLA-G, and/or negative for CD117. In a specific embodiment, said stemcell or population of placental stem cells is isolated away fromplacental cells that are not stem cells. In another specific embodiment,said isolated placental stem cell is non-maternal in origin. In anotherspecific embodiment, at least about 90%, at least about 95%, or at leastabout 99%, of said cells in said isolated population of placental stemcells, are non-maternal in origin. In another specific embodiment, saidstem cell or population of placental stem cells is isolated away fromplacental stem cells that do not display these characteristics. Inanother embodiment, provided herein is a method of obtaining a placentalstem cell that is HLA A,B,C⁻, CD45⁻, CD34⁻, CD133⁻ and positive forCD10, CD13, CD38, CD44, CD90, CD105, CD200 and/or HLA-G, and/or negativefor CD117, comprising isolating said cell from placental perfusate.

In another embodiment, provided herein is a placental stem cell that isCD200⁺ and CD10⁺, as determined by antibody binding, and CD117⁻, asdetermined by both antibody binding and RT-PCR. In another embodiment,provided herein is a placental stem cell that is CD10⁺, CD29⁻, CD54⁺,CD200⁺, HLA-G⁺, HLA class I⁻ and β-2-microglobulin⁻. In anotherembodiment, provided herein are placental stem cells, wherein theexpression of at least one marker is at least two-fold higher than for amesenchymal stem cell (e.g., a bone marrow-derived mesenchymal stemcell). In another specific embodiment, said isolated placental stem cellis non-maternal in origin. In another specific embodiment, at leastabout 90%, at least about 95%, or at least 99%, of said cells in saidisolated population of placental stem cells, are non-maternal in origin.

In another embodiment, provided herein is an isolated population ofplacental stem cells, wherein a plurality of said placental stem cellsare positive for aldehyde dehydrogenase (ALDH), as assessed by analdehyde dehydrogenase activity assay. Such assays are known in the art(see, e.g., Bostian and Betts, Biochem. J., 173, 787, (1978)). In aspecific embodiment, said ALDH assay uses ALDEFLUOR® (Aldagen, Inc.,Ashland, Oreg.) as a marker of aldehyde dehydrogenase activity. In aspecific embodiment, said plurality is between about 3% and about 25% ofcells in said population of cells. In another embodiment, providedherein is a population of umbilical cord stem cells, wherein a pluralityof said umbilical cord stem cells are positive for aldehydedehydrogenase, as assessed by an aldehyde dehydrogenase activity assaythat uses ALDEFLUOR® as an indicator of aldehyde dehydrogenase activity.In a specific embodiment, said plurality is between about 3% and about25% of cells in said population of cells. In another embodiment, saidpopulation of placental stem cells or umbilical cord stem cells shows atleast three-fold, or at least five-fold, higher ALDH activity than apopulation of bone marrow-derived mesenchymal stem cells having the samenumber of cells and cultured under the same conditions.

For any of the above placental stem cells, or populations of placentalstem cells, the stem cell or population of placental stem cells are, orcan comprise, cells that have been passaged at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, 18, or 20 times, or more, or expanded for 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38 or 40 population doublings, or more.

In a specific embodiment of any of the above placental cells or cellpopulations, the karyotype of the cells, or at least about 95% or about99% of the cells in said population, is normal. In another specificembodiment of any of the above placental cells or cell populations, thecells, or cells in the population of cells, are non-maternal in origin.

Isolated placental stem cells, or isolated populations of placental stemcells, bearing any of the above combinations of markers, can be combinedin any ratio. Any two or more of the above placental stem cellpopulations can be isolated, or enriched, to form a placental stem cellpopulation. For example, an isolated population of placental stem cellscomprising a first population of placental stem cells defined by one ofthe marker combinations described above can be combined with a secondpopulation of placental stem cells defined by another of the markercombinations described above, wherein said first and second populationsare combined in a ratio of about 1:99, 2:98, 3:97, 4:96, 5:95, 10:90,20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4,97:3, 98:2, or about 99:1. In like fashion, any three, four, five ormore of the above-described placental stem cells or placental stem cellpopulations can be combined.

In a specific embodiment of the above-mentioned placental stem cells,the placental stem cells constitutively secrete IL-6, IL-8 and monocytechemoattractant protein (MCP-1).

The immunosuppressive pluralities of placental stem cells describedabove can comprise about, at least, or no more than, 1×10⁵, 5×10⁵,1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰,1×10¹¹ or more placental stem cells.

5.2.3 Selecting and Producing Placental Stem Cell Populations

In another embodiment, provided herein is a method of selecting aplurality of immunosuppressive placental stem cells from a plurality ofplacental cells, comprising selecting a population of placental cellswherein at least 10%, at least 20%, at least 30%, at least 40%, at least50% at least 60%, at least 70%, at least 80%, at least 90%, or at least95% of said cells are CD200⁺, HLA-G⁺ placental stem cells, and whereinsaid placental stem cells detectably suppresses T cell proliferation ina mixed lymphocyte reaction (MLR) assay. In a specific embodiment, saidselecting comprises selecting stem cells that are also CD73⁺ and CD105⁺.In another specific embodiment, said selecting comprises selecting stemcells that are also CD34⁻, CD38⁻ or CD45⁻. In another specificembodiment, said selecting comprises selecting placental stem cells thatare also CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In another specificembodiment, said selecting also comprises selecting a plurality ofplacental stem cells that forms one or more embryoid-like bodies whencultured under conditions that allow the formation of embryoid-likebodies.

In another embodiment, provided herein is a method of selecting aplurality of immunosuppressive placental stem cells from a plurality ofplacental cells, comprising selecting a plurality of placental cellswherein at least 10%, at least 20%, at least 30%, at least 40%, at least50% at least 60%, at least 70%, at least 80%, at least 90%, or at least95% of said cells are CD73⁺, CD105⁺, CD200⁺ placental stem cells, andwherein said placental stem cells detectably suppresses T cellproliferation in a mixed lymphocyte reaction (MLR) assay. In a specificembodiment, said selecting comprises selecting stem cells that are alsoHLA-G⁺. In another specific embodiment, said selecting comprisesselecting placental stem cells that are also CD34⁻, CD38⁻ or CD45⁻. Inanother specific embodiment, said selecting comprises selectingplacental stem cells that are also CD34⁻, CD38⁻ and CD45⁻. In anotherspecific embodiment, said selecting comprises selecting placental stemcells that are also CD34⁻, CD38⁻, CD45⁻, and HLA-G⁺. In another specificembodiment, said selecting additionally comprises selecting a populationof placental cells that produces one or more embryoid-like bodies whenthe population is cultured under conditions that allow the formation ofembryoid-like bodies.

In another embodiment, also provided herein is a method of selecting aplurality of immunosuppressive placental stem cells from a plurality ofplacental cells, comprising selecting a plurality of placental cellswherein at least 10%, at least 20%, at least 30%, at least 40%, at least50% at least 60%, at least 70%, at least 80%, at least 90%, or at least95% of said cells are CD200⁺, OCT-4⁺ placental stem cells, and whereinsaid placental stem cells detectably suppresses T cell proliferation ina mixed lymphocyte reaction (MLR) assay. In a specific embodiment, saidselecting comprises selecting placental stem cells that are also CD73⁺and CD105⁺. In another specific embodiment, said selecting comprisesselecting placental stem cells that are also HLA-G⁺. In another specificembodiment, said selecting comprises selecting placental stem cells thatare also CD34⁻, CD38⁻ and CD45⁻. In another specific embodiment, saidselecting comprises selecting placental stem cells that are also CD34⁻,CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁺.

In another embodiment, provided herein is a method of selecting aplurality of immunosuppressive placental stem cells from a plurality ofplacental cells, comprising selecting a plurality of placental cellswherein at least 10%, at least 20%, at least 30%, at least 40%, at least50% at least 60%, at least 70%, at least 80%, at least 90%, or at least95% of said cells are CD73⁺, CD105⁺ and HLA-G⁺ placental stem cells, andwherein said placental stem cells detectably suppresses T cellproliferation in a mixed lymphocyte reaction (MLR) assay. In a specificembodiment, said selecting comprises selecting placental stem cells thatare also CD34⁻, CD38⁻ or CD45⁻. In another specific embodiment, saidselecting comprises selecting placental stem cells that are also CD34⁻,CD38⁻ and CD45⁻. In another specific embodiment, said selectingcomprises selecting placental stem cells that are also CD200⁺. Inanother specific embodiment, said selecting comprises selectingplacental stem cells that are also CD34⁻, CD38⁻, CD45⁻, OCT-4⁺ andCD200⁺.

In another embodiment, also provided herein is provides a method ofselecting a plurality of immunosuppressive placental stem cells from aplurality of placental cells, comprising selecting a plurality ofplacental cells wherein at least 10%, at least 20%, at least 30%, atleast 40%, at least 50% at least 60%, at least 70%, at least 80%, atleast 90%, or at least 95% of said cells are CD73⁺, CD105⁺ placentalstem cells, and wherein said plurality forms one or more embryoid-likebodies under conditions that allow formation of embryoid-like bodies. Ina specific embodiment, said selecting comprises selecting placental stemcells that are also CD34⁻, CD38⁻ or CD45⁻ In another specificembodiment, said selecting comprises selecting placental stem cells thatare also CD34⁻, CD38⁻ and CD45⁻. In another specific embodiment, saidselecting comprises selecting placental stem cells that are also OCT-4⁺.In a more specific embodiment, said selecting comprises selectingplacental stem cells that are also OCT-4⁺, CD34⁻, CD38⁻ and CD45⁻.

In another embodiment, provided herein is a method of selecting aplurality of immunosuppressive placental stem cells from a plurality ofplacental cells, comprising selecting a plurality of placental cellswherein at least 10%, at least 20%, at least 30%, at least 40%, at least50% at least 60%, at least 70%, at least 80%, at least 90%, or at least95% of said isolated placental cells are OCT4⁺ stem cells, and whereinsaid plurality forms one or more embryoid-like bodies under conditionsthat allow formation of embryoid-like bodies. In a specific embodiment,said selecting comprises selecting placental stem cells that are alsoCD73⁺ and CD105⁺. In another specific embodiment, said selectingcomprises selecting placental stem cells that are also CD34⁻, CD38⁻, orCD45⁻. In another specific embodiment, said selecting comprisesselecting placental stem cells that are also CD200⁺. In a more specificembodiment, said selecting comprises selecting placental stem cells thatare also CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻, and CD45⁻.

Immunosuppressive populations, or pluralities, of placental stem cellscan be produced according to the methods provided herein. For example,provided herein is method of producing a cell population, comprisingselecting any of the pluralities of placental stem cells describedabove, and isolating the plurality of placental stem cells from othercells, e.g., other placental cells. In a specific embodiment, providedherein is a method of producing a cell population comprising selectingplacental cells, wherein said placental cells (a) adhere to a substrate,(b) express CD200 and HLA-G, or express CD73, CD105, and CD200, orexpress CD200 and OCT-4, or express CD73, CD105, and HLA-G, or expressCD73 and CD105 and facilitate the formation of one or more embryoid-likebodies in a population of placental cells that comprise the stem cell,when said population is cultured under conditions that allow formationof embryoid-like bodies, or express OCT-4 and facilitate the formationof one or more embryoid-like bodies in a population of placental cellsthat comprise the stem cell, when said population is cultured underconditions that allow formation of embryoid-like bodies; and (c)detectably suppress CD4⁺ or CD8⁺ T cell proliferation in an MLR (mixedlymphocyte reaction) or regression assay; and isolating said placentalcells from other cells to form a cell population.

In a more specific embodiment, immunosuppressive placental stem cellpopulations can be produced by a method comprising selecting placentalstem cells that (a) adhere to a substrate, (b) express CD200 and HLA-G,and (c) detectably suppress CD4⁺ or CD8⁺ T cell proliferation in an MLR(mixed lymphocyte reaction); and isolating said placental stem cellsfrom other cells to form a cell population. In another specificembodiment, the method comprises selecting placental stem cells that (a)adhere to a substrate, (b) express CD73, CD105, and CD200, and (c)detectably suppress CD4⁺ or CD8⁺ T cell proliferation in an MLR; andisolating said placental stem cells from other cells to form a cellpopulation. In another specific embodiment, provided herein is a methodof producing a cell population comprising selecting placental stem cellsthat (a) adhere to a substrate, (b) express CD200 and OCT-4, and (c)detectably suppress CD4⁺ or CD8⁺ T cell proliferation in an MLR; andisolating said placental stem cells from other cells to form a cellpopulation. In another specific embodiment, provided herein is a methodof producing a cell population comprising selecting placental stem cellsthat (a) adhere to a substrate, (b) express CD73 and CD105, (c) formembryoid-like bodies when cultured under conditions allowing theformation of embryoid-like bodies, and (d) detectably suppress CD4⁺ orCD8⁺ T cell proliferation in an MLR; and isolating said placental stemcells from other cells to form a cell population. In another specificembodiment, the method comprises selecting placental stem cells that (a)adhere to a substrate, (b) express CD73, CD105, and (c) detectablysuppress CD4⁺ or CD8⁺ T cell proliferation in an MLR; and isolating saidplacental stem cells from other cells to form a cell population. Amethod of producing a cell population comprising selecting placentalstem cells that (a) adhere to a substrate, (b) express OCT-4, (c) formembryoid-like bodies when cultured under conditions allowing theformation of embryoid-like bodies, and (d) detectably suppress CD4⁺ orCD8⁺ T cell proliferation in an MLR; and isolating said placental stemcells from other cells to form a cell population.

In a specific embodiment of the methods of producing animmunosuppressive placental stem cell population, said T cells and saidplacental cells are present in said MLR at a ratio of about 5:1. Theplacental cells used in the method can be derived from the wholeplacenta, or primarily from amnion, or amnion and chorion. In anotherspecific embodiment, the placental cells suppress CD4⁺ or CD8⁺ T cellproliferation by at least 50%, at least 75%, at least 90%, or at least95% in said MLR compared to an amount of T cell proliferation in saidMLR in the absence of said placental cells. The method can additionallycomprise the selection and/or production of a placental stem cellpopulation capable of immunomodulation, e.g., suppression of theactivity of, other immune cells, e.g., an activity of a natural killer(NK) cell.

5.2.4 Growth in Culture

The growth of the placental stem cells described herein, as for anymammalian cell, depends in part upon the particular medium selected forgrowth. Under optimum conditions, placental stem cells typically doublein number in 3-5 days. During culture, the placental stem cells providedherein adhere to a substrate in culture, e.g. the surface of a tissueculture container (e.g., tissue culture dish plastic, fibronectin-coatedplastic, and the like) and form a monolayer.

Populations of isolated placental cells that comprise the placental stemcells provided herein, when cultured under appropriate conditions, formembryoid-like bodies, that is, three-dimensional clusters of cells growatop the adherent stem cell layer. Cells within the embryoid-like bodiesexpress markers associated with very early stem cells, e.g., OCT-4,Nanog, SSEA3 and SSEA4. Cells within the embryoid-like bodies aretypically not adherent to the culture substrate, as are the placentalstem cells described herein, but remain attached to the adherent cellsduring culture. Embryoid-like body cells are dependent upon the adherentplacental stem cells for viability, as embryoid-like bodies do not formin the absence of the adherent stem cells. The adherent placental stemcells thus facilitate the growth of one or more embryoid-like bodies ina population of placental cells that comprise the adherent placentalstem cells. Without wishing to be bound by theory, the cells of theembryoid-like bodies are thought to grow on the adherent placental stemcells much as embryonic stem cells grow on a feeder layer of cells.Mesenchymal stem cells, e.g., bone marrow-derived mesenchymal stemcells, do not develop embryoid-like bodies in culture.

5.2.5 Differentiation

The placental stem cells, useful in the methods of treating diseases,disorder or conditions associated with, or caused by inappropriate orharmful immune response, e.g., inflammation, provided herein, aredifferentiable into different committed cell lineages. For example, theplacental stem cells can be differentiated into cells of an adipogenic,chondrogenic, neurogenic, or osteogenic lineage. Such differentiationcan be accomplished, e.g., by any method known in the art fordifferentiating, e.g., bone marrow-derived mesenchymal stem cells intosimilar cell lineages, or by methods described elsewhere herein.

The placental stem cells and umbilical cord stem cells provided hereincan exhibit the capacity to differentiate into a particular cell lineagein vitro, in vivo, or in vitro and in vivo. In a specific embodiment,the placental stem cells and umbilical cord stem cells provided hereincan be differentiated in vitro when placed in conditions that cause orpromote differentiation into a particular cell lineage, but do notdetectably differentiate in vivo, e.g., in a NOD-SCID mouse model.

5.3 Methods of Obtaining Placental Stem Cells 5.3.1 Stem Cell CollectionComposition

Placental stem cells can be collected and isolated according to themethods provided herein. Generally, stem cells are obtained from amammalian placenta using a physiologically-acceptable solution, e.g., astem cell collection composition. A stem cell collection composition isdescribed in detail in related U.S. Provisional Application No.60/754,969, entitled “Improved Composition for Collecting and PreservingPlacental Stem Cells and Methods of Using the Composition” filed on Dec.29, 2005.

The stem cell collection composition can comprise anyphysiologically-acceptable solution suitable for the collection and/orculture of stem cells, for example, a saline solution (e.g.,phosphate-buffered saline, Kreb's solution, modified Kreb's solution,Eagle's solution, 0.9% NaCl. etc.), a culture medium (e.g., DMEM, HDMEM,etc.), and the like.

The stem cell collection composition can comprise one or more componentsthat tend to preserve placental stem cells, that is, prevent theplacental stem cells from dying, or delay the death of the placentalstem cells, reduce the number of placental stem cells in a population ofcells that die, or the like, from the time of collection to the time ofculturing. Such components can be, e.g., an apoptosis inhibitor (e.g., acaspase inhibitor or JNK inhibitor); a vasodilator (e.g., magnesiumsulfate, an antihypertensive drug, atrial natriuretic peptide (ANP),adrenocorticotropin, corticotropin-releasing hormone, sodiumnitroprusside, hydralazine, adenosine triphosphate, adenosine,indomethacin or magnesium sulfate, a phosphodiesterase inhibitor, etc.);a necrosis inhibitor (e.g., 2-(1H-Indol-3-yl)-3-pentylamino-maleimide,pyrrolidine dithiocarbamate, or clonazepam); a TNF-α inhibitor; and/oran oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromide,perfluorodecyl bromide, etc.).

The stem cell collection composition can comprise one or moretissue-degrading enzymes, e.g., a metalloprotease, a serine protease, aneutral protease, an RNase, or a DNase, or the like. Such enzymesinclude, but are not limited to, collagenases (e.g., collagenase I, II,III or IV, a collagenase from Clostridium histolyticum, etc.); dispase,thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like.

The stem cell collection composition can comprise a bacteriocidally orbacteriostatically effective amount of an antibiotic. In certainnon-limiting embodiments, the antibiotic is a macrolide (e.g.,tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime,cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, anerythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g.,ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, astreptomycin, etc. In a particular embodiment, the antibiotic is activeagainst Gram(+) and/or Gram(−) bacteria, e.g., Pseudomonas aeruginosa,Staphylococcus aureus, and the like.

The stem cell collection composition can also comprise one or more ofthe following compounds: adenosine (about 1 mM to about 50 mM);D-glucose (about 20 mM to about 100 mM); magnesium ions (about 1 mM toabout 50 mM); a macromolecule of molecular weight greater than 20,000daltons, in one embodiment, present in an amount sufficient to maintainendothelial integrity and cellular viability (e.g., a synthetic ornaturally occurring colloid, a polysaccharide such as dextran or apolyethylene glycol present at about 25 g/l to about 100 g/1, or about40 g/l to about 60 g/1); an antioxidant (e.g., butylated hydroxyanisole,butylated hydroxytoluene, glutathione, vitamin C or vitamin E present atabout 25 μM to about 100 μM); a reducing agent (e.g., N-acetylcysteinepresent at about 0.1 mM to about 5 mM); an agent that prevents calciumentry into cells (e.g., verapamil present at about 2 μM to about 25 μM);nitroglycerin (e.g., about 0.05 g/L to about 0.2 g/L); an anticoagulant,in one embodiment, present in an amount sufficient to help preventclotting of residual blood (e.g., heparin or hirudin present at aconcentration of about 1000 units/1 to about 100,000 units/1); or anamiloride containing compound (e.g., amiloride, ethyl isopropylamiloride, hexamethylene amiloride, dimethyl amiloride or isobutylamiloride present at about 1.0 μM to about 5 μM).

5.3.2 Collection and Handling of Placenta

Generally, a human placenta is recovered shortly after its expulsionafter birth. In a preferred embodiment, the placenta is recovered from apatient after informed consent and after a complete medical history ofthe patient is taken and is associated with the placenta. Preferably,the medical history continues after delivery. Such a medical history canbe used to coordinate subsequent use of the placenta or the stem cellsharvested therefrom. For example, human placental stem cells can beused, in light of the medical history, for personalized medicine for theinfant associated with the placenta, or for parents, siblings or otherrelatives of the infant.

Prior to recovery of placental stem cells, the umbilical cord blood andplacental blood are removed. In certain embodiments, after delivery, thecord blood in the placenta is recovered. The placenta can be subjectedto a conventional cord blood recovery process. Typically a needle orcannula is used, with the aid of gravity, to exsanguinate the placenta(see, e.g., Anderson, U.S. Pat. No. 5,372,581; Hessel et al., U.S. Pat.No. 5,415,665). The needle or cannula is usually placed in the umbilicalvein and the placenta can be gently massaged to aid in draining cordblood from the placenta. Such cord blood recovery may be performedcommercially, e.g., LifeBank Inc., Cedar Knolls, N.J., ViaCord, CordBlood Registry and Cryocell. Preferably, the placenta is gravity drainedwithout further manipulation so as to minimize tissue disruption duringcord blood recovery.

Typically, a placenta is transported from the delivery or birthing roomto another location, e.g., a laboratory, for recovery of cord blood andcollection of stem cells by, e.g., perfusion or tissue dissociation. Theplacenta is preferably transported in a sterile, thermally insulatedtransport device (maintaining the temperature of the placenta between20-28° C.), for example, by placing the placenta, with clamped proximalumbilical cord, in a sterile zip-lock plastic bag, which is then placedin an insulated container. In another embodiment, the placenta istransported in a cord blood collection kit substantially as described inpending U.S. patent application Ser. No. 11/230,760, filed Sep. 19,2005. Preferably, the placenta is delivered to the laboratory four totwenty-four hours following delivery. In certain embodiments, theproximal umbilical cord is clamped, preferably within 4-5 cm(centimeter) of the insertion into the placental disc prior to cordblood recovery. In other embodiments, the proximal umbilical cord isclamped after cord blood recovery but prior to further processing of theplacenta.

The placenta, prior to stem cell collection, can be stored under sterileconditions and at either room temperature or at a temperature of 5 to25° C. (centigrade). The placenta may be stored for a period of longerthan forty eight hours, and preferably for a period of four totwenty-four hours prior to perfusing the placenta to remove any residualcord blood. The placenta is preferably stored in an anticoagulantsolution at a temperature of 5 to 25° C. (centigrade). Suitableanticoagulant solutions are well known in the art. For example, asolution of heparin or warfarin sodium can be used. In a preferredembodiment, the anticoagulant solution comprises a solution of heparin(e.g., 1% w/w in 1:1000 solution). The exsanguinated placenta ispreferably stored for no more than 36 hours before placental stem cellsare collected.

The mammalian placenta or a part thereof, once collected and preparedgenerally as above, can be treated in any art-known manner, e.g., can beperfused or disrupted, e.g., digested with one or more tissue-disruptingenzymes, to obtain stem cells.

5.3.3 Physical Disruption and Enzymatic Digestion of Placental Tissue

In one embodiment, stem cells are collected from a mammalian placenta byphysical disruption, e.g., enzymatic digestion, of the organ, e.g.,using the stem cell collection composition described in Section 5.3.1,above. For example, the placenta, or a portion thereof, may be, e.g.,crushed, sheared, minced, diced, chopped, macerated or the like, whilein contact with, e.g., a buffer, medium or a stem cell collectioncomposition, and the tissue subsequently digested with one or moreenzymes. The placenta, or a portion thereof, may also be physicallydisrupted and digested with one or more enzymes, and the resultingmaterial then immersed in, or mixed into, a buffer, medium or a stemcell collection composition. Any method of physical disruption can beused, provided that the method of disruption leaves a plurality, morepreferably a majority, and more preferably at least 60%, 70%, 80%, 90%,95%, 98%, or 99% of the cells in said organ viable, as determined by,e.g., trypan blue exclusion.

The placenta can be dissected into components prior to physicaldisruption and/or enzymatic digestion and stem cell recovery. Forexample, placental stem cells can be obtained from the amnioticmembrane, chorion, placental cotyledons, or any combination thereof, orumbilical cord, or any combination thereof. Preferably, placental stemcells are obtained from placental tissue comprising amnion and chorion,or amnion-chorion and umbilical cord. In one embodiment, stem cells areobtained from amnion-chorion and umbilical cord in about a 1:1 weightratio. Typically, placental stem cells can be obtained by disruption ofa small block of placental tissue, e.g., a block of placental tissuethat is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or about 1000 cubicmillimeters in volume.

A preferred stem cell collection composition comprises one or moretissue-disruptive enzyme(s). Enzymatic digestion preferably uses acombination of enzymes, e.g., a combination of a matrix metalloproteaseand a neutral protease, for example, a combination of collagenase anddispase. In one embodiment, enzymatic digestion of placental tissue usesa combination of a matrix metalloprotease, a neutral protease, and amucolytic enzyme for digestion of hyaluronic acid, such as a combinationof collagenase, dispase, and hyaluronidase or a combination of LIBERASE(Boehringer Mannheim Corp., Indianapolis, Ind.) and hyaluronidase. Otherenzymes that can be used to disrupt placenta tissue include papain,deoxyribonucleases, serine proteases, such as trypsin, chymotrypsin, orelastase. Serine proteases may be inhibited by alpha 2 microglobulin inserum and therefore the medium used for digestion is usually serum-free.EDTA and DNase are commonly used in enzyme digestion procedures toincrease the efficiency of cell recovery. The digestate is preferablydiluted so as to avoid trapping stem cells within the viscous digest.

Any combination of tissue digestion enzymes can be used. Typicalconcentrations for tissue digestion enzymes include, e.g., 50-200 U/mLfor collagenase I and collagenase IV, 1-10 U/mL for dispase, and 10-100U/mL for elastase. Proteases can be used in combination, that is, two ormore proteases in the same digestion reaction, or can be usedsequentially in order to liberate placental stem cells. For example, inone embodiment, a placenta, or part thereof, is digested first with anappropriate amount of collagenase I at 2 mg/ml for 30 minutes, followedby digestion with trypsin, 0.25%, for 10 minutes, at 37° C. Serineproteases are preferably used consecutively following use of otherenzymes.

In another embodiment, the tissue can further be disrupted by theaddition of a chelator, e.g., ethylene glycol bis(2-aminoethylether)-N,N,N′N′-tetraacetic acid (EGTA) or ethylenediaminetetraaceticacid (EDTA) to the stem cell collection composition comprising the stemcells, or to a solution in which the tissue is disrupted and/or digestedprior to isolation of the stem cells with the stem cell collectioncomposition.

It will be appreciated that where an entire placenta, or portion of aplacenta comprising both fetal and maternal cells (for example, wherethe portion of the placenta comprises the chorion or cotyledons), theplacental stem cells collected will comprise a mix of placental stemcells derived from both fetal and maternal sources. Where a portion ofthe placenta that comprises no, or a negligible number of, maternalcells (for example, amnion), the placental stem cells collected willcomprise almost exclusively fetal placental stem cells.

5.3.4 Placental Perfusion

Placental stem cells can also be obtained by perfusion of the mammalianplacenta. Methods of perfusing mammalian placenta to obtain stem cellsare disclosed, e.g., in Hariri, U.S. Application Publication No.2002/0123141, and in related U.S. Provisional Application No.60/754,969, entitled “Improved Composition for Collecting and PreservingPlacental Stem Cells and Methods of Using the Composition” filed on Dec.29, 2005.

Placental stem cells can be collected by perfusion, e.g., through theplacental vasculature, using, e.g., a stem cell collection compositionas a perfusion solution. In one embodiment, a mammalian placenta isperfused by passage of perfusion solution through either or both of theumbilical artery and umbilical vein. The flow of perfusion solutionthrough the placenta may be accomplished using, e.g., gravity flow intothe placenta. Preferably, the perfusion solution is forced through theplacenta using a pump, e.g., a peristaltic pump. The umbilical vein canbe, e.g., cannulated with a cannula, e.g., a TEFLON® or plastic cannula,that is connected to a sterile connection apparatus, such as steriletubing. The sterile connection apparatus is connected to a perfusionmanifold.

In preparation for perfusion, the placenta is preferably oriented (e.g.,suspended) in such a manner that the umbilical artery and umbilical veinare located at the highest point of the placenta. The placenta can beperfused by passage of a perfusion fluid, e.g., the stem cell collectioncomposition provided herein, through the placental vasculature, orthrough the placental vasculature and surrounding tissue. In oneembodiment, the umbilical artery and the umbilical vein are connectedsimultaneously to a pipette that is connected via a flexible connectorto a reservoir of the perfusion solution. The perfusion solution ispassed into the umbilical vein and artery. The perfusion solution exudesfrom and/or passes through the walls of the blood vessels into thesurrounding tissues of the placenta, and is collected in a suitable openvessel from the surface of the placenta that was attached to the uterusof the mother during gestation. The perfusion solution may also beintroduced through the umbilical cord opening and allowed to flow orpercolate out of openings in the wall of the placenta which interfacedwith the maternal uterine wall. In another embodiment, the perfusionsolution is passed through the umbilical veins and collected from theumbilical artery, or is passed through the umbilical artery andcollected from the umbilical veins.

In one embodiment, the proximal umbilical cord is clamped duringperfusion, and more preferably, is clamped within 4-5 cm (centimeter) ofthe cord's insertion into the placental disc.

The first collection of perfusion fluid from a mammalian placenta duringthe exsanguination process is generally colored with residual red bloodcells of the cord blood and/or placental blood. The perfusion fluidbecomes more colorless as perfusion proceeds and the residual cord bloodcells are washed out of the placenta. Generally from 30 to 100 ml(milliliter) of perfusion fluid is adequate to initially exsanguinatethe placenta, but more or less perfusion fluid may be used depending onthe observed results.

The volume of perfusion liquid used to collect placental stem cells mayvary depending upon the number of stem cells to be collected, the sizeof the placenta, the number of collections to be made from a singleplacenta, etc. In various embodiments, the volume of perfusion liquidmay be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mLto 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL.Typically, the placenta is perfused with 700-800 mL of perfusion liquidfollowing exsanguination.

The placenta can be perfused a plurality of times over the course ofseveral hours or several days. Where the placenta is to be perfused aplurality of times, it may be maintained or cultured under asepticconditions in a container or other suitable vessel, and perfused withthe stem cell collection composition, or a standard perfusion solution(e.g., a normal saline solution such as phosphate buffered saline(“PBS”)) with or without an anticoagulant (e.g., heparin, warfarinsodium, coumarin, bishydroxycoumarin), and/or with or without anantimicrobial agent (e.g., β-mercaptoethanol (0.1 mM); antibiotics suchas streptomycin (e.g., at 40-100 μg/ml), penicillin (e.g., at 40 U/ml),amphotericin B (e.g., at 0.5 μg/ml). In one embodiment, an isolatedplacenta is maintained or cultured for a period of time withoutcollecting the perfusate, such that the placenta is maintained orcultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3 or more days beforeperfusion and collection of perfusate. The perfused placenta can bemaintained for one or more additional time(s), e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 ormore hours, and perfused a second time with, e.g., 700-800 mL perfusionfluid. The placenta can be perfused 1, 2, 3, 4, 5 or more times, forexample, once every 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment,perfusion of the placenta and collection of perfusion solution, e.g.,stem cell collection composition, is repeated until the number ofrecovered nucleated cells falls below 100 cells/ml. The perfusates atdifferent time points can be further processed individually to recovertime-dependent populations of cells, e.g., stem cells. Perfusates fromdifferent time points can also be pooled.

Without wishing to be bound by any theory, after exsanguination and asufficient time of perfusion of the placenta, placental stem cells arebelieved to migrate into the exsanguinated and perfused microcirculationof the placenta where they are collected, preferably by washing into acollecting vessel by perfusion. Perfusing the isolated placenta not onlyserves to remove residual cord blood but also provide the placenta withthe appropriate nutrients, including oxygen. The placenta may becultivated and perfused with a similar solution which was used to removethe residual cord blood cells, preferably, without the addition ofanticoagulant agents.

Perfusion as described herein results in the collection of significantlymore placental stem cells than the number obtainable from a mammalianplacenta not perfused with said solution, and not otherwise treated toobtain stem cells (e.g., by tissue disruption, e.g., enzymaticdigestion). In this context, “significantly more” means at least 10%more. Perfusion yields significantly more placental stem cells than,e.g., the number of placental stem cells obtainable from culture mediumin which a placenta, or portion thereof, has been cultured.

Stem cells can be isolated from placenta by perfusion with a solutioncomprising one or more proteases or other tissue-disruptive enzymes. Ina specific embodiment, a placenta or portion thereof (e.g., amnioticmembrane, amnion and chorion, placental lobule or cotyledon, orcombination of any of the foregoing) is brought to 25-37° C., and isincubated with one or more tissue-disruptive enzymes in 200 mL of aculture medium for 30 minutes. Cells from the perfusate are collected,brought to 4° C., and washed with a cold inhibitor mix comprising 5 mMEDTA, 2 mM dithiothreitol and 2 mM beta-mercaptoethanol. The stem cellsare washed after several minutes with a cold (e.g., 4° C.) stem cellcollection composition described elsewhere herein.

It will be appreciated that perfusion using the pan method, that is,whereby perfusate is collected after it has exuded from the maternalside of the placenta, results in a mix of fetal and maternal cells. As aresult, the cells collected by this method comprise a mixed populationof placental stem cells of both fetal and maternal origin. In contrast,perfusion solely through the placental vasculature, whereby perfusionfluid is passed through one or two placental vessels and is collectedsolely through the remaining vessel(s), results in the collection of apopulation of placental stem cells almost exclusively of fetal origin.

5.3.5 Isolation, Sorting, and Characterization of Placental Stem Cells

Stem cells from mammalian placenta, whether obtained by perfusion orenzymatic digestion, can initially be purified from (i.e., be isolatedfrom) other cells by Ficoll gradient centrifugation. Such centrifugationcan follow any standard protocol for centrifugation speed, etc. In oneembodiment, for example, cells collected from the placenta are recoveredfrom perfusate by centrifugation at 5000×g for 15 minutes at roomtemperature, which separates cells from, e.g., contaminating debris andplatelets. In another embodiment, placental perfusate is concentrated toabout 200 ml, gently layered over Ficoll, and centrifuged at about1100×g for 20 minutes at 22° C., and the low-density interface layer ofcells is collected for further processing.

Cell pellets can be resuspended in fresh stem cell collectioncomposition, or a medium suitable for stem cell maintenance, e.g., IMDMserum-free medium containing 2 U/ml heparin and 2 mM EDTA (GibcoBRL,NY). The total mononuclear cell fraction can be isolated, e.g., usingLymphoprep (Nycomed Pharma, Oslo, Norway) according to themanufacturer's recommended procedure.

As used herein, “isolating” placental stem cells means to remove atleast 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the cellswith which the stem cells are normally associated in the intactmammalian placenta. A stem cell from an organ is “isolated” when it ispresent in a population of cells that comprises fewer than 50% of thecells with which the stem cell is normally associated in the intactorgan.

Placental cells obtained by perfusion or digestion can, for example, befurther, or initially, isolated by differential trypsinization using,e.g., a solution of 0.05% trypsin with 0.2% EDTA (Sigma, St. Louis Mo.).Differential trypsinization is possible because placental stem cellstypically detach from plastic surfaces within about five minutes whereasother adherent populations typically require more than 20-30 minutesincubation. The detached placental stem cells can be harvested followingtrypsinization and trypsin neutralization, using, e.g., TrypsinNeutralizing Solution (TNS, Cambrex). In one embodiment of isolation ofadherent cells, aliquots of, for example, about 5-10×10⁶ cells areplaced in each of several T-75 flasks, preferably fibronectin-coated T75flasks. In such an embodiment, the cells can be cultured withcommercially available Mesenchymal Stem Cell Growth Medium (MSCGM)(Cambrex), and placed in a tissue culture incubator (37° C., 5% CO₂).After 10 to 15 days, non-adherent cells are removed from the flasks bywashing with PBS. The PBS is then replaced by MSCGM. Flasks arepreferably examined daily for the presence of various adherent celltypes and in particular, for identification and expansion of clusters offibroblastoid cells.

The number and type of cells collected from a mammalian placenta can bemonitored, for example, by measuring changes in morphology and cellsurface markers using standard cell detection techniques such as flowcytometry, cell sorting, immunocytochemistry (e.g., staining with tissuespecific or cell-marker specific antibodies) fluorescence activated cellsorting (FACS), magnetic activated cell sorting (MACS), by examinationof the morphology of cells using light or confocal microscopy, and/or bymeasuring changes in gene expression using techniques well known in theart, such as PCR and gene expression profiling. These techniques can beused, too, to identify cells that are positive for one or moreparticular markers. For example, using antibodies to CD34, one candetermine, using the techniques above, whether a cell comprises adetectable amount of CD34; if so, the cell is CD34⁺. Likewise, if a cellproduces enough OCT-4 RNA to be detectable by RT-PCR, or significantlymore OCT-4 RNA than an adult cell, the cell is OCT-4⁺ Antibodies to cellsurface markers (e.g., CD markers such as CD34) and the sequence of stemcell-specific genes, such as OCT-4, are well-known in the art.

Placental cells, particularly cells that have been isolated by Ficollseparation, differential adherence, or a combination of both, may besorted using a fluorescence activated cell sorter (FACS). Fluorescenceactivated cell sorting (FACS) is a well-known method for separatingparticles, including cells, based on the fluorescent properties of theparticles (Kamarch, 1987, Methods Enzymol, 151:150-165). Laserexcitation of fluorescent moieties in the individual particles resultsin a small electrical charge allowing electromagnetic separation ofpositive and negative particles from a mixture. In one embodiment, cellsurface marker-specific antibodies or ligands are labeled with distinctfluorescent labels. Cells are processed through the cell sorter,allowing separation of cells based on their ability to bind to theantibodies used. FACS sorted particles may be directly deposited intoindividual wells of 96-well or 384-well plates to facilitate separationand cloning.

In one sorting scheme, stem cells from placenta are sorted on the basisof expression of the markers CD34, CD38, CD44, CD45, CD73, CD105, OCT-4and/or HLA-G. This can be accomplished in connection with procedures toselect stem cells on the basis of their adherence properties in culture.For example, an adherence selection stem can be accomplished before orafter sorting on the basis of marker expression. In one embodiment, forexample, cells are sorted first on the basis of their expression ofCD34; CD34⁻ cells are retained, and cells that are CD200⁺ HLA-G⁺, areseparated from all other CD34⁻ cells. In another embodiment, cells fromplacenta are based on their expression of markers CD200 and/or HLA-G;for example, cells displaying either of these markers are isolated forfurther use. Cells that express, e.g., CD200 and/or HLA-G can, in aspecific embodiment, be further sorted based on their expression of CD73and/or CD105, or epitopes recognized by antibodies SH2, SH3 or SH4, orlack of expression of CD34, CD38 or CD45. For example, in oneembodiment, placental cells are sorted by expression, or lack thereof,of CD200, HLA-G, CD73, CD105, CD34, CD38 and CD45, and placental cellsthat are CD200⁺, HLA-G⁺, CD73⁺, CD105⁺, CD34⁻, CD38⁻ and CD45⁻ areisolated from other placental cells for further use.

In another embodiment, magnetic beads can be used to separate cells. Thecells may be sorted using a magnetic activated cell sorting (MACS)technique, a method for separating particles based on their ability tobind magnetic beads (0.5-100 μm diameter). A variety of usefulmodifications can be performed on the magnetic microspheres, includingcovalent addition of antibody that specifically recognizes a particularcell surface molecule or hapten. The beads are then mixed with the cellsto allow binding. Cells are then passed through a magnetic field toseparate out cells having the specific cell surface marker. In oneembodiment, these cells can then isolated and re-mixed with magneticbeads coupled to an antibody against additional cell surface markers.The cells are again passed through a magnetic field, isolating cellsthat bound both the antibodies. Such cells can then be diluted intoseparate dishes, such as microtiter dishes for clonal isolation.

Placental stem cells can also be characterized and/or sorted based oncell morphology and growth characteristics. For example, placental stemcells can be characterized as having, and/or selected on the basis of,e.g., a fibroblastoid appearance in culture. Placental stem cells canalso be characterized as having, and/or be selected, on the basis oftheir ability to form embryoid-like bodies. In one embodiment, forexample, placental cells that are fibroblastoid in shape, express CD73and CD105, and produce one or more embryoid-like bodies in culture areisolated from other placental cells. In another embodiment, OCT-4⁺placental cells that produce one or more embryoid-like bodies in cultureare isolated from other placental cells.

In another embodiment, placental stem cells can be identified andcharacterized by a colony forming unit assay. Colony forming unit assaysare commonly known in the art, such as Mesen Cult™ medium (Stem CellTechnologies, Inc., Vancouver British Columbia)

Placental stem cells can be assessed for viability, proliferationpotential, and longevity using standard techniques known in the art,such as trypan blue exclusion assay, fluorescein diacetate uptake assay,propidium iodide uptake assay (to assess viability); and thymidineuptake assay, MTT cell proliferation assay (to assess proliferation).Longevity may be determined by methods well known in the art, such as bydetermining the maximum number of population doubling in an extendedculture.

Placental stem cells can also be separated from other placental cellsusing other techniques known in the art, e.g., selective growth ofdesired cells (positive selection), selective destruction of unwantedcells (negative selection); separation based upon differential cellagglutinability in the mixed population as, for example, with soybeanagglutinin; freeze-thaw procedures; filtration; conventional and zonalcentrifugation; centrifugal elutriation (counter-streamingcentrifugation); unit gravity separation; countercurrent distribution;electrophoresis; and the like.

5.4 Culture of Placental Stem Cells 5.4.1 Culture Media

Isolated placental stem cells, or placental stem cell population, orcells or placental tissue from which placental stem cells grow out, canbe used to initiate, or seed, cell cultures. Cells are generallytransferred to sterile tissue culture vessels either uncoated or coatedwith extracellular matrix or ligands such as laminin, collagen (e.g.,native or denatured), gelatin, fibronectin, ornithine, vitronectin, andextracellular membrane protein (e.g., MATRIGEL (BD Discovery Labware,Bedford, Mass.)).

Placental stem cells can be cultured in any medium, and under anyconditions, recognized in the art as acceptable for the culture of stemcells. Preferably, the culture medium comprises serum. Placental stemcells can be cultured in, for example, DMEM-LG (Dulbecco's ModifiedEssential Medium, low glucose)/MCDB 201 (chick fibroblast basal medium)containing ITS (insulin-transferrin-selenium), LA+BSA (linoleicacid-bovine serum albumin), dextrose, L-ascorbic acid, PDGF, EGF, IGF-1,and penicillin/streptomycin; DMEM-HG (high glucose) comprising 10% fetalbovine serum (FBS); DMEM-HG comprising 15% FBS; IMDM (Iscove's modifiedDulbecco's medium) comprising 10% FBS, 10% horse serum, andhydrocortisone; M199 comprising 10% FBS, EGF, and heparin; α-MEM(minimal essential medium) comprising 10% FBS, GlutaMAX™ and gentamicin;DMEM comprising 10% FBS, GlutaMAX™ and gentamicin, etc. A preferredmedium is DMEM-LG/MCDB-201 comprising 2% FBS, ITS, LA+BSA, dextrose,L-ascorbic acid, PDGF, EGF, and penicillin/streptomycin.

Other media in that can be used to culture placental stem cells includeDMEM (high or low glucose), Eagle's basal medium, Ham's F10 medium(F10), Ham's F-12 medium (F12), Iscove's modified Dulbecco's medium,Mesenchymal Stem Cell Growth Medium (MSCGM), Liebovitz's L-15 medium,MCDB, DMIEM/F12, RPMI 1640, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma),and CELL-GRO FREE.

The culture medium can be supplemented with one or more componentsincluding, for example, serum (e.g., fetal bovine serum (FBS),preferably about 2-15% (v/v); equine (horse) serum (ES); human serum(HS)); beta-mercaptoethanol (BME), preferably about 0.001% (v/v); one ormore growth factors, for example, platelet-derived growth factor (PDGF),epidermal growth factor (EGF), basic fibroblast growth factor (bFGF),insulin-like growth factor-1 (IGF-1), leukemia inhibitory factor (LIF),vascular endothelial growth factor (VEGF), and erythropoietin (EPO);amino acids, including L-valine; and one or more antibiotic and/orantimycotic agents to control microbial contamination, such as, forexample, penicillin G, streptomycin sulfate, amphotericin B, gentamicin,and nystatin, either alone or in combination.

5.4.2 Expansion and Proliferation of Placental Stem Cells

Once an isolated placental stem cell, or isolated population of stemcells (e.g., a stem cell or population of stem cells separated from atleast 50% of the placental cells with which the stem cell or populationof stem cells is normally associated in vivo), the stem cell orpopulation of stem cells can be proliferated and expanded in vitro. Forexample, a population of placental stem cells can be cultured in tissueculture containers, e.g., dishes, flasks, multiwell plates, or the like,for a sufficient time for the stem cells to proliferate to 70-90%confluence, that is, until the stem cells and their progeny occupy70-90% of the culturing surface area of the tissue culture container.

Placental stem cells can be seeded in culture vessels at a density thatallows cell growth. For example, the cells may be seeded at low density(e.g., about 1,000 to about 5,000 cells/cm²) to high density (e.g.,about 50,000 or more cells/cm²). In a preferred embodiment, the cellsare cultured at about 0 to about 5 percent by volume CO₂ in air. In somepreferred embodiments, the cells are cultured at about 2 to about 25percent O₂ in air, preferably about 5 to about 20 percent O₂ in air. Thecells preferably are cultured at about 25° C. to about 40° C.,preferably 37° C. The cells are preferably cultured in an incubator. Theculture medium can be static or agitated, for example, using abioreactor. Placental stem cells preferably are grown under lowoxidative stress (e.g., with addition of glutathione, ascorbic acid,catalase, tocopherol, N-acetylcysteine, or the like).

Once 70%-90% confluence is obtained, the cells may be passaged. Forexample, the cells can be enzymatically treated, e.g., trypsinized,using techniques well-known in the art, to separate them from the tissueculture surface. After removing the cells by pipetting and counting thecells, about 20,000-100,000 stem cells, preferably about 50,000 stemcells, are passaged to a new culture container containing fresh culturemedium. Typically, the new medium is the same type of medium from whichthe stem cells were removed. Provided herein are populations ofplacental stem cells that have been passaged at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, 18, or 20 times, or more, and combinations ofthe same.

5.4.3 Placental Stem Cell Populations

The methods of treatment provided herein, in certain embodiments, usepopulations of placental stem cells. Placental stem cell populations canbe isolated directly from one or more placentas; that is, the placentalstem cell population can be a population of placental cells, comprisingplacental stem cells, obtained from, or contained within, perfusate, orobtained from, or contained within, digestate (that is, the collectionof cells obtained by enzymatic digestion of a placenta or part thereof).Isolated placental stem cells as described herein can also be culturedand expanded to produce placental stem cell populations. Populations ofplacental cells comprising placental stem cells can also be cultured andexpanded to produce placental stem cell populations.

Placental stem cell populations described herein comprise placental stemcells, for example, placental stem cells as described herein. In variousembodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,or 99% of the cells in an isolated placental stem cell population areplacental stem cells. That is, a placental stem cell population cancomprise, e.g., as much as 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% non-stem cells.

Provided herein are methods of producing isolated placental stem cellpopulation by, e.g., selecting placental stem cells, whether derivedfrom enzymatic digestion or perfusion, that express particular markersand/or particular culture or morphological characteristics. In oneembodiment, for example, a cell population can be produced by a methodcomprising selecting placental cells that (a) adhere to a substrate, and(b) express CD200 and HLA-G; and isolating said cells from other cellsto form a cell population. In another embodiment, the method ofproducing a cell population comprises selecting placental cells that (a)adhere to a substrate, and (b) express CD73, CD105, and CD200; andisolating said cells from other cells to form a cell population. Inanother embodiment, the method of producing a cell population comprisesselecting placental cells that (a) adhere to a substrate and (b) expressCD200 and OCT-4; and isolating said cells from other cells to form acell population. In another embodiment, the method of producing a cellpopulation comprises selecting placental cells that (a) adhere to asubstrate, (b) express CD73 and CD105, and (c) facilitate the formationof one or more embryoid-like bodies in a population of placental cellscomprising said stem cell when said population is cultured underconditions that allow for the formation of an embryoid-like body; andisolating said cells from other cells to form a cell population. Inanother embodiment, the method of producing a cell population comprisesselecting placental cells that (a) adhere to a substrate, and (b)express CD73, CD105 and HLA-G; and isolating said cells from other cellsto form a cell population. In another embodiment, the method ofproducing a cell population comprises selecting placental cells that (a)adhere to a substrate, (b) express OCT-4, and (c) facilitate theformation of one or more embryoid-like bodies in a population ofplacental cells comprising said stem cell when said population iscultured under conditions that allow for the formation of anembryoid-like body; and isolating said cells from other cells to form acell population. In any of the above embodiments, the method canadditionally comprise selecting placental cells that express ABC-p (aplacenta-specific ABC transporter protein; see, e.g., Allikmets et al.,Cancer Res. 58(23):5337-9 (1998)). The method can also compriseselecting cells exhibiting at least one characteristic specific to,e.g., a mesenchymal stem cell, for example, expression of CD29,expression of CD44, expression of CD90, or expression of a combinationof the foregoing.

In the above embodiments, the substrate can be any surface on whichculture and/or selection of cells, e.g., placental stem cells, can beaccomplished. Typically, the substrate is plastic, e.g., tissue culturedish or multiwell plate plastic. Tissue culture plastic can be coatedwith a biomolecule, e.g., laminin or fibronectin.

Cells, e.g., placental stem cells, can be selected for a placental stemcell population by any means known in the art of cell selection. Forexample, cells can be selected using an antibody or antibodies to one ormore cell surface markers, for example, in flow cytometry or FACS.Selection can be accomplished using antibodies in conjunction withmagnetic beads. Antibodies that are specific for certain stemcell-related markers are known in the art. For example, antibodies toOCT-4 (Abcam, Cambridge, Mass.), CD200 (Abcam), HLA-G (Abcam), CD73 (BDBiosciences Pharmingen, San Diego, Calif.), CD105 (Abcam; BioDesignInternational, Saco, Me.), etc. Antibodies to other markers are alsoavailable commercially, e.g., CD34, CD38 and CD45 are available from,e.g., StemCell Technologies or BioDesign International.

The isolated placental stem cell population can comprise placental cellsthat are not stem cells, or cells that are not placental cells.

Isolated placental stem cell populations can be combined with one ormore populations of non-stem cells or non-placental cells. For example,an isolated population of placental stem cells can be combined withblood (e.g., placental blood or umbilical cord blood), blood-derivedstem cells (e.g., stem cells derived from placental blood or umbilicalcord blood), populations of blood-derived nucleated cells, bonemarrow-derived mesenchymal cells, bone-derived stem cell populations,crude bone marrow, adult (somatic) stem cells, populations of stem cellscontained within tissue, cultured stem cells, populations offully-differentiated cells (e.g., chondrocytes, fibroblasts, amnioticcells, osteoblasts, muscle cells, cardiac cells, etc.) and the like.Cells in an isolated placental stem cell population can be combined witha plurality of cells of another type in ratios of about 100,000,000:1,50,000,000:1, 20,000,000:1, 10,000,000:1, 5,000,000:1, 2,000,000:1,1,000,000:1, 500,000:1, 200,000:1, 100,000:1, 50,000:1, 20,000:1,10,000:1, 5,000:1, 2,000:1, 1,000:1, 500:1, 200:1, 100:1, 50:1, 20:1,10:1, 5:1, 2:1, 1:1; 1:2; 1:5; 1:10; 1:100; 1:200; 1:500; 1:1,000;1:2,000; 1:5,000; 1:10,000; 1:20,000; 1:50,000; 1:100,000; 1:500,000;1:1,000,000; 1:2,000,000; 1:5,000,000; 1:10,000,000; 1:20,000,000;1:50,000,000; or about 1:100,000,000, comparing numbers of totalnucleated cells in each population. Cells in an isolated placental stemcell population can be combined with a plurality of cells of a pluralityof cell types, as well.

In one, an isolated population of placental stem cells is combined witha plurality of hematopoietic stem cells. Such hematopoietic stem cellscan be, for example, contained within unprocessed placental, umbilicalcord blood or peripheral blood; in total nucleated cells from placentalblood, umbilical cord blood or peripheral blood; in an isolatedpopulation of CD34⁺ cells from placental blood, umbilical cord blood orperipheral blood; in unprocessed bone marrow; in total nucleated cellsfrom bone marrow; in an isolated population of CD34⁺ cells from bonemarrow, or the like.

5.5 Preservation of Placental Stem Cells

Placental stem cells can be preserved, that is, placed under conditionsthat allow for long-term storage, or conditions that inhibit cell deathby, e.g., apoptosis or necrosis.

Placental stem cells can be preserved using, e.g., a compositioncomprising an apoptosis inhibitor, necrosis inhibitor and/or anoxygen-carrying perfluorocarbon, as described in related U.S.Provisional Application No. 60/754,969, entitled “Improved Compositionfor Collecting and Preserving Placental Stem Cells and Methods of Usingthe Composition” filed on Dec. 25, 2005. In one embodiment, providedherein is a method of preserving a population of stem cells comprisingcontacting said population of stem cells with a stem cell collectioncomposition comprising an inhibitor of apoptosis and an oxygen-carryingperfluorocarbon, wherein said inhibitor of apoptosis is present in anamount and for a time sufficient to reduce or prevent apoptosis in thepopulation of stem cells, as compared to a population of stem cells notcontacted with the inhibitor of apoptosis. In a specific embodiment,said inhibitor of apoptosis is a caspase inhibitor. In another specificembodiment, said inhibitor of apoptosis is a JNK inhibitor. In a morespecific embodiment, said JNK inhibitor does not modulatedifferentiation or proliferation of said stem cells. In anotherembodiment, said stem cell collection composition comprises saidinhibitor of apoptosis and said oxygen-carrying perfluorocarbon inseparate phases. In another embodiment, said stem cell collectioncomposition comprises said inhibitor of apoptosis and saidoxygen-carrying perfluorocarbon in an emulsion. In another embodiment,the stem cell collection composition additionally comprises anemulsifier, e.g., lecithin. In another embodiment, said apoptosisinhibitor and said perfluorocarbon are between about 0° C. and about 25°C. at the time of contacting the stem cells. In another more specificembodiment, said apoptosis inhibitor and said perfluorocarbon arebetween about 2° C. and 10° C., or between about 2° C. and about 5° C.,at the time of contacting the stem cells. In another more specificembodiment, said contacting is performed during transport of saidpopulation of stem cells. In another more specific embodiment, saidcontacting is performed during freezing and thawing of said populationof stem cells.

In another embodiment, populations of placental stem cells can bepreserved by a method comprising contacting said population of stemcells with an inhibitor of apoptosis and an organ-preserving compound,wherein said inhibitor of apoptosis is present in an amount and for atime sufficient to reduce or prevent apoptosis in the population of stemcells, as compared to a population of stem cells not contacted with theinhibitor of apoptosis. In a specific embodiment, the organ-preservingcompound is UW solution (described in U.S. Pat. No. 4,798,824; alsoknown as ViaSpan; see also Southard et al., Transplantation49(2):251-257 (1990)) or a solution described in Stern et al., U.S. Pat.No. 5,552,267. In another embodiment, said organ-preserving compound ishydroxyethyl starch, lactobionic acid, raffinose, or a combinationthereof. In another embodiment, the stem cell collection compositionadditionally comprises an oxygen-carrying perfluorocarbon, either in twophases or as an emulsion.

In another embodiment of the method, placental stem cells are contactedwith a stem cell collection composition comprising an apoptosisinhibitor and oxygen-carrying perfluorocarbon, organ-preservingcompound, or combination thereof, during perfusion. In anotherembodiment, said stem cells are contacted during a process of tissuedisruption, e.g., enzymatic digestion. In another embodiment, placentalstem cells are contacted with said stem cell collection compound aftercollection by perfusion, or after collection by tissue disruption, e.g.,enzymatic digestion.

Typically, during placental cell collection, enrichment and isolation,it is preferable to minimize or eliminate cell stress due to hypoxia andmechanical stress. In another embodiment of the method, therefore, astem cell, or population of stem cells, is exposed to a hypoxiccondition during collection, enrichment or isolation for less than sixhours during said preservation, wherein a hypoxic condition is aconcentration of oxygen that is less than normal blood oxygenconcentration. In a more specific embodiment, said population of stemcells is exposed to said hypoxic condition for less than two hoursduring said preservation. In another more specific embodiment, saidpopulation of stem cells is exposed to said hypoxic condition for lessthan one hour, or less than thirty minutes, or is not exposed to ahypoxic condition, during collection, enrichment or isolation. Inanother specific embodiment, said population of stem cells is notexposed to shear stress during collection, enrichment or isolation.

The placental stem cells described herein can be cryopreserved, e.g., incryopreservation medium in small containers, e.g., ampoules. Suitablecryopreservation medium includes, but is not limited to, culture mediumincluding, e.g., growth medium, or cell freezing medium, for examplecommercially available cell freezing medium, e.g., C2695, C2639 or C6039(Sigma). Cryopreservation medium preferably comprises DMSO(dimethylsulfoxide), at a concentration of, e.g., about 10% (v/v).Cryopreservation medium may comprise additional agents, for example,Plasmalyte, methylcellulose and/or glycerol. Placental stem cells arepreferably cooled at about 1° C./min during cryopreservation. Apreferred cryopreservation temperature is about −80° C. to about −180°C., preferably about −125° C. to about −140° C. Cryopreserved cells canbe transferred to liquid nitrogen prior to thawing for use. In someembodiments, for example, once the ampoules have reached about −90° C.,they are transferred to a liquid nitrogen storage area. Cryopreservedcells preferably are thawed at a temperature of about 25° C. to about40° C., preferably to a temperature of about 37° C.

5.6 Uses of Placental Stem Cells 5.6.1 Compositions Comprising PlacentalStem Cells

The methods of immunosuppression provided herein can use compositionscomprising placental stem cells, or biomolecules therefrom. In the samemanner, the pluralities and populations of placental stem cells providedherein can be combined with any physiologically-acceptable ormedically-acceptable compound, composition or device for use in, e.g.,research or therapeutics.

5.6.1.1 Cryopreserved Placental Stem Cells

The immunosuppressive placental stem cells, and populations of thecells, described herein can be preserved, for example, cryopreserved forlater use. Methods for cryopreservation of cells, such as stem cells,are well known in the art. Placental stem cell populations can beprepared in a form that is easily administrable to an individual. Forexample, placental stem cells, or populations of the placental stemcells, described herein can be contained within a container that issuitable for medical use. Such a container can be, for example, asterile plastic bag, flask, jar, or other container from which theplacental stem cell population can be easily dispensed. For example, thecontainer can be a blood bag or other plastic, medically-acceptable bagsuitable for the intravenous administration of a liquid to a recipient.The container is preferably one that allows for cryopreservation of thecombined stem cell population.

Cryopreserved immunosuppressive placental stem cell populations cancomprise placental stem cells derived from a single donor, or frommultiple donors. The placental stem cell population can be completelyHLA-matched to an intended recipient, or partially or completelyHLA-mismatched.

Thus, in one embodiment, provided herein is a composition comprising animmunosuppressive placental stem cell population in a container. In aspecific embodiment, the stem cell population is cryopreserved. Inanother specific embodiment, the container is a bag, flask, or jar. Inmore specific embodiment, said bag is a sterile plastic bag. In a morespecific embodiment, said bag is suitable for, allows or facilitatesintravenous administration of said placental stem cell population. Thebag can comprise multiple lumens or compartments that are interconnectedto allow mixing of the placental stem cells and one or more othersolutions, e.g., a drug, prior to, or during, administration. In anotherspecific embodiment, the composition comprises one or more compoundsthat facilitate cryopreservation of the combined stem cell population.In another specific embodiment, said placental stem cell population iscontained within a physiologically-acceptable aqueous solution. In amore specific embodiment, said physiologically-acceptable aqueoussolution is a 0.9% NaCl solution. In another specific embodiment, saidplacental stem cell population comprises placental cells that areHLA-matched to a recipient of said stem cell population. In anotherspecific embodiment, said combined stem cell population comprisesplacental cells that are at least partially HLA-mismatched to arecipient of said stem cell population. In another specific embodiment,said placental stem cells are derived from a plurality of donors.

5.6.1.2 Pharmaceutical Compositions

Immunosuppressive populations of placental stem cells, or populations ofcells comprising placental stem cells, can be formulated intopharmaceutical compositions for use in vivo. Such pharmaceuticalcompositions comprise a population of placental stem cells, or apopulation of cells comprising placental stem cells, in apharmaceutically-acceptable carrier, e.g., a saline solution or otheraccepted physiologically-acceptable solution for in vivo administration.Pharmaceutical compositions provided herein can comprise any of theplacental stem cell populations, or placental stem cell types, describedelsewhere herein. The pharmaceutical compositions can comprise fetal,maternal, or both fetal and maternal placental stem cells. Thepharmaceutical compositions provided herein can further compriseplacental stem cells obtained from a single individual or placenta, orfrom a plurality of individuals or placentae.

The pharmaceutical compositions provided herein can comprise anyimmunosuppressive number of placental stem cells. For example, a singleunit dose of placental stem cells can comprise, in various embodiments,about, at least, or no more than 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷,5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹ or moreplacental stem cells.

The pharmaceutical compositions provided herein can comprise populationsof cells that comprise 50% viable cells or more (that is, at least 50%of the cells in the population are functional or living). Preferably, atleast 60% of the cells in the population are viable. More preferably, atleast 70%, 80%, 90%, 95%, or 99% of the cells in the population in thepharmaceutical composition are viable.

The pharmaceutical compositions provided herein can comprise one or morecompounds that, e.g., facilitate engraftment (e.g., anti-T-cell receptorantibodies, an immunosuppressant, or the like); stabilizers such asalbumin, dextran 40, gelatin, hydroxyethyl starch, and the like.

5.6.1.3 Placental Stem Cell Conditioned Media

The placental stem cells provided herein can be used to produceconditioned medium that is immunosuppressive, that is, medium comprisingone or more biomolecules secreted or excreted by the stem cells thathave a detectable immunosuppressive effect on a plurality of one or moretypes of immune cells. In various embodiments, the conditioned mediumcomprises medium in which placental stem cells have grown for at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. In otherembodiments, the conditioned medium comprises medium in which placentalstem cells have grown to at least 30%, 40%, 50%, 60%, 70%, 80%, 90%confluence, or up to 100% confluence. Such conditioned medium can beused to support the culture of a separate population of placental stemcells, or stem cells of another kind. In another embodiment, theconditioned medium comprises medium in which placental stem cells havebeen differentiated into an adult cell type. In another embodiment, theconditioned medium comprises medium in which placental stem cells andnon-placental stem cells have been cultured.

Thus, in one embodiment, provided herein is a composition comprisingculture medium from a culture of placental stem cells, wherein saidplacental stem cells (a) adhere to a substrate; (b) express CD200 andHLA-G, or express CD73, CD105, and CD200, or express CD200 and OCT-4, orexpress CD73, CD105, and HLA-G, or express CD73 and CD105 and facilitatethe formation of one or more embryoid-like bodies in a population ofplacental cells that comprise the placental stem cells, when saidpopulation is cultured under conditions that allow formation ofembryoid-like bodies, or express OCT-4 and facilitate the formation ofone or more embryoid-like bodies in a population of placental cells thatcomprise the placental stem cells when said population is cultured underconditions that allow formation of embryoid-like bodies; and (c)detectably suppress CD4⁺ or CD8⁺ T cell proliferation in an MLR (mixedlymphocyte reaction), wherein said culture of placental stem cells hasbeen cultured in said medium for 24 hours or more. In a specificembodiment, the composition further comprises a plurality of saidplacental stem cells. In another specific embodiment, the compositioncomprises a plurality of non-placental cells. In a more specificembodiment, said non-placental cells comprise CD34⁺ cells, e.g.,hematopoietic progenitor cells, such as peripheral blood hematopoieticprogenitor cells, cord blood hematopoietic progenitor cells, orplacental blood hematopoietic progenitor cells. The non-placental cellscan also comprise other stem cells, such as mesenchymal stem cells,e.g., bone marrow-derived mesenchymal stem cells. The non-placentalcells can also be one ore more types of adult cells or cell lines. Inanother specific embodiment, the composition comprises ananti-proliferative agent, e.g., an anti-MIP-1α or anti-MIP-1β antibody.

5.6.1.4 Matrices Comprising Placental Stem Cells

Further provided herein are matrices, hydrogels, scaffolds, and the likethat comprise immunosuppressive placental stem cells, e.g., animmunosuppressive population of placental stem cells.

Placental stem cells provided herein can be seeded onto a naturalmatrix, e.g., a placental biomaterial such as an amniotic membranematerial. Such an amniotic membrane material can be, e.g., amnioticmembrane dissected directly from a mammalian placenta; fixed orheat-treated amniotic membrane, substantially dry (i.e., <20% H₂O)amniotic membrane, chorionic membrane, substantially dry chorionicmembrane, substantially dry amniotic and chorionic membrane, and thelike. Preferred placental biomaterials on which placental stem cells canbe seeded are described in Hariri, U.S. Application Publication No.2004/0048796.

Placental stem cells provided herein can be suspended in a hydrogelsolution suitable for, e.g., injection. Suitable hydrogels for suchcompositions include self-assembling peptides, such as RAD16. In oneembodiment, a hydrogel solution comprising the cells can be allowed toharden, for instance in a mold, to form a matrix having cells dispersedtherein for implantation. Placental stem cells in such a matrix can alsobe cultured so that the cells are mitotically expanded prior toimplantation. The hydrogel is, e.g., an organic polymer (natural orsynthetic) that is cross-linked via covalent, ionic, or hydrogen bondsto create a three-dimensional open-lattice structure that entraps watermolecules to form a gel. Hydrogel-forming materials includepolysaccharides such as alginate and salts thereof, peptides,polyphosphazines, and polyacrylates, which are crosslinked ionically, orblock polymers such as polyethylene oxide-polypropylene glycol blockcopolymers which are crosslinked by temperature or pH, respectively. Insome embodiments, the hydrogel or matrix is biodegradable.

In some embodiments, the formulation comprises an in situ polymerizablegel (see, e.g., U.S. Patent Application Publication 2002/0022676; Ansethet al., J. Control Release, 78(1-3):199-209 (2002); Wang et al.,Biomaterials, 24(22):3969-80 (2003).

In some embodiments, the polymers are at least partially soluble inaqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof. Examples of polymers having acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

The placental stem cells or co-cultures thereof can be seeded onto athree-dimensional framework or scaffold and implanted in vivo. Such aframework can be implanted in combination with any one or more growthfactors, cells, drugs or other components that stimulate tissueformation or otherwise enhance or improve the practice of the methods oftreatment described elsewhere herein.

Examples of scaffolds that can be used in the methods of treatmentdescribed herein include nonwoven mats, porous foams, or self assemblingpeptides. Nonwoven mats can be formed using fibers comprised of asynthetic absorbable copolymer of glycolic and lactic acids (e.g.,PGA/PLA) (VICRYL, Ethicon, Inc., Somerville, N.J.). Foams, composed of,e.g., poly(ε-caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer,formed by processes such as freeze-drying, or lyophilization (see, e.g.,U.S. Pat. No. 6,355,699), can also be used as scaffolds.

In another embodiment, the scaffold is, or comprises, a nanofibrousscaffold, e.g., an electrospun nanofibrous scaffold. In a more specificembodiment, said nanofibrous scaffold comprises poly(L-lactic acid)(PLLA), type I collagen, a copolymer of vinylidene fluoride andtrifluoroethylene (PVDF-TrFE), poly(-caprolactone),poly(L-lactide-co-ε-caprolactone) [P(LLA-CL)] (e.g., 75:25), and/or acopolymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) andtype I collagen. In another more specific embodiment, said scaffoldpromotes the differentiation of placental stem cells into chondrocytes.Methods of producing nanofibrous scaffolds, e.g., electrospunnanofibrous scaffolds, are known in the art. See, e.g., Xu et al.,Tissue Engineering 10(7): 1160-1168 (2004); Xu et al., Biomaterials25:877-886 (20040; Meng et al., J. Biomaterials Sci., Polymer Edition18(1):81-94 (2007).

Placental stem cells described herein, e.g., immunosuppressive placentalstem cells, can also be seeded onto, or contacted with, aphysiologically-acceptable ceramic material including, but not limitedto, mono-, di-, tri-, alpha-tri-, beta-tri-, and tetra-calciumphosphate, hydroxyapatite, fluoroapatites, calcium sulfates, calciumfluorides, calcium oxides, calcium carbonates, magnesium calciumphosphates, biologically active glasses such as BIOGLASS®, and mixturesthereof. Porous biocompatible ceramic materials currently commerciallyavailable include SURGIBONE® (CanMedica Corp., Canada), ENDOBON® (MerckBiomaterial France, France), CEROS® (Mathys, AG, Bettlach, Switzerland),and mineralized collagen bone grafting products such as HEALOS™ (DePuy,Inc., Raynham, Mass.) and VITOSS®, RHAKOSS™, and CORTOSS® (Orthovita,Malvern, Pa.). The framework can be a mixture, blend or composite ofnatural and/or synthetic materials.

In another embodiment, placental stem cells can be seeded onto, orcontacted with, a felt, which can be, e.g., composed of a multifilamentyarn made from a bioabsorbable material such as PGA, PLA, PCL copolymersor blends, or hyaluronic acid.

The placental stem cells described herein can, in another embodiment, beseeded onto foam scaffolds that may be composite structures. Such foamscaffolds can be molded into a useful shape, such as that of a portionof a specific structure in the body to be repaired, replaced oraugmented. In some embodiments, the framework is treated, e.g., with0.1M acetic acid followed by incubation in polylysine, PBS, and/orcollagen, prior to inoculation of the immunosuppressive placental stemcells in order to enhance cell attachment. External surfaces of a matrixmay be modified to improve the attachment or growth of cells anddifferentiation of tissue, such as by plasma-coating the matrix, oraddition of one or more proteins (e.g., collagens, elastic fibers,reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparinsulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate,keratin sulfate, etc.), a cellular matrix, and/or other materials suchas, but not limited to, gelatin, alginates, agar, agarose, and plantgums, and the like.

In some embodiments, the scaffold comprises, or is treated with,materials that render it non-thrombogenic. These treatments andmaterials may also promote and sustain endothelial growth, migration,and extracellular matrix deposition. Examples of these materials andtreatments include but are not limited to natural materials such asbasement membrane proteins such as laminin and Type IV collagen,synthetic materials such as EPTFE, and segmented polyurethaneureasilicones, such as PURSPAN™ (The Polymer Technology Group, Inc.,Berkeley, Calif.). The scaffold can also comprise anti-thrombotic agentssuch as heparin; the scaffolds can also be treated to alter the surfacecharge (e.g., coating with plasma) prior to seeding with placental stemcells.

5.6.2 Genetically Modified Placental Stem Cells

In another aspect, provided herein are placental stem cells andumbilical cord stem cells that are genetically modified, e.g., toproduce a nucleic acid or polypeptide of interest. Genetic modificationcan be accomplished, e.g., using virus-based vectors including, but notlimited to, non-integrating replicating vectors, e.g., papilloma virusvectors, SV40 vectors, adenoviral vectors; integrating viral vectors,e.g., retrovirus vector or adeno-associated viral vectors; orreplication-defective viral vectors. Other methods of introducing DNAinto cells include the use of liposomes, electroporation, a particlegun, direct DNA injection, or the like.

Stem cells can be, e.g., transformed or transfected with DNA controlledby or in operative association with, one or more appropriate expressioncontrol elements, for example, promoter or enhancer sequences,transcription terminators, polyadenylation sites, internal ribosomalentry sites. Preferably, such a DNA incorporates a selectable marker.Following the introduction of the foreign DNA, engineered stem cells canbe, e.g., grown in enriched media and then switched to selective media.In one embodiment, the DNA used to engineer a placental stem cellcomprises a nucleotide sequence encoding a polypeptide of interest,e.g., a cytokine, growth factor, differentiation agent, or therapeuticpolypeptide.

The DNA used to engineer the stem cell can comprise any promoter knownin the art to drive expression of a nucleotide sequence in mammaliancells, e.g., human cells. For example, promoters include, but are notlimited to, CMV promoter/enhancer, SV40 promoter, papillomaviruspromoter, Epstein-Barr virus promoter, elastin gene promoter, and thelike. In a specific embodiment, the promoter is regulatable so that thenucleotide sequence is expressed only when desired. Promoters can beeither inducible (e.g., those associated with metallothionein and heatshock proteins) or constitutive.

In another specific embodiment, the promoter is tissue-specific orexhibits tissue specificity. Examples of such promoters include but arenot limited to: myelin basic protein gene control region (Readhead etal., 1987, Cell 48:703) (oligodendrocyte cells); elastase I gene controlregion (Swit et al., 1984, Cell 38:639; Ornitz et al., 1986, Cold SpringHarbor Symp. Quant. Biol. 50:399; MacDonald, 1987, Hepatology 7:425)(pancreatic acinar cells); insulin gene control region (Hanahan, 1985,Nature 315:115) (pancreatic beta cells); myosin light chain-2 genecontrol region (Shani, 1985, Nature 314:283) (skeletal muscle).

Placental stem cells may be engineered to “knock out” or “knock down”expression of one or more genes. The expression of a gene native to acell can be diminished by, for example, inhibition of expression byinactivating the gene completely by, e.g., homologous recombination. Inone embodiment, for example, an exon encoding an important region of theprotein, or an exon 5′ to that region, is interrupted by a positiveselectable marker, e.g., neo, preventing the production of normal mRNAfrom the target gene and resulting in inactivation of the gene. A genemay also be inactivated by creating a deletion in part of a gene or bydeleting the entire gene. By using a construct with two regions ofhomology to the target gene that are far apart in the genome, thesequences intervening the two regions can be deleted (Mombaerts et al.,1991, Proc. Nat. Acad. Sci. U.S.A. 88:3084). Antisense, DNAzymes, smallinterfering RNA, and ribozyme molecules that inhibit expression of thetarget gene can also be used to reduce the level of target gene activityin the stem cells. For example, antisense RNA molecules which inhibitthe expression of major histocompatibility gene complexes (HLA) havebeen shown to be most versatile with respect to immune responses. Triplehelix molecules can be utilized in reducing the level of target geneactivity. See, e.g., L. G. Davis et al. (eds), 1994, BASIC METHODS INMOLECULAR BIOLOGY, 2nd ed., Appleton & Lange, Norwalk, Conn., which isincorporated herein by reference.

In a specific embodiment, placental or umbilical cord stem cells can begenetically modified with a nucleic acid molecule comprising anucleotide sequence encoding a polypeptide of interest, whereinexpression of the polypeptide of interest is controllable by anexogenous factor, e.g., polypeptide, small organic molecule, or thelike. Such a polypeptide can be a therapeutic polypeptide. In a morespecific embodiment, the polypeptide of interest is IL-12 orinterleukin-1 receptor antagonist (IL-1Ra). In another more specificembodiment, the polypeptide of interest is a fusion of interleukin-1receptor antagonist and dihydrofolate reductase (DHFR), and theexogenous factor is an antifolate, e.g., methotrexate. Such a constructis useful in the engineering of placental or umbilical cord stem cellsthat express IL-1Ra, or a fusion of IL-1Ra and DHFR, upon contact withmethotrexate. Such a construct can be used, e.g., in the treatment ofrheumatoid arthritis. In this embodiment, the fusion of IL-1Ra and DHFRis translationally upregulated upon exposure to an antifolate such asmethotrexate. Therefore, in another specific embodiment, the nucleicacid used to genetically engineer a placental stem cell or umbilicalcord stem cell can comprise nucleotide sequences encoding a firstpolypeptide and a second polypeptide, wherein said first and secondpolypeptides are expressed as a fusion protein that is translationallyupregulated in the presence of an exogenous factor. The polypeptide canbe expressed transiently or long-term (e.g., over the course of weeks ormonths).

Such a nucleic acid molecule can additionally comprise a nucleotidesequence encoding a polypeptide that allows for positive selection ofengineered stem cells, or allows for visualization of the engineeredstem cells. In another more specific embodiment, the nucleotide sequenceencodes a polypeptide that is, e.g., fluorescent under appropriatevisualization conditions, e.g., luciferase (Luc). In a more specificembodiment, such a nucleic acid molecule can compriseIL-1Ra-DHFR-IRES-Luc, where IRES is an internal ribosomal entry site.

5.6.3 Immortalized Placental Stem Cell Lines

Mammalian placental cells can be conditionally immortalized bytransfection with any suitable vector containing a growth-promotinggene, that is, a gene encoding a protein that, under appropriateconditions, promotes growth of the transfected cell, such that theproduction and/or activity of the growth-promoting protein isregulatable by an external factor. In a preferred embodiment thegrowth-promoting gene is an oncogene such as, but not limited to, v-myc,N-myc, c-myc, p53, SV40 large T antigen, polyoma large T antigen, Elaadenovirus or E7 protein of human papillomavirus.

External regulation of the growth-promoting protein can be achieved byplacing the growth-promoting gene under the control of anexternally-regulatable promoter, e.g., a promoter the activity of whichcan be controlled by, for example, modifying the temperature of thetransfected cells or the composition of the medium in contact with thecells. in one embodiment, a tetracycline (tet)-controlled geneexpression system can be employed (see Gossen et al., Proc. Natl. Acad.Sci. USA 89:5547-5551, 1992; Hoshimaru et al., Proc. Natl. Acad. Sci.USA 93:1518-1523, 1996). In the absence of tet, a tet-controlledtransactivator (tTA) within this vector strongly activates transcriptionfrom ph_(CMV*-1), a minimal promoter from human cytomegalovirus fused totet operator sequences. tTA is a fusion protein of the repressor (tetR)of the transposon-10-derived tet resistance operon of Escherichia coliand the acidic domain of VP16 of herpes simplex virus. Low, non-toxicconcentrations of tet (e.g., 0.01-1.0 μg/mL) almost completely abolishtransactivation by tTA.

In one embodiment, the vector further contains a gene encoding aselectable marker, e.g., a protein that confers drug resistance. Thebacterial neomycin resistance gene (neo^(R)) is one such marker that maybe employed within the methods described herein. Cells carrying neo^(R)may be selected by means known to those of ordinary skill in the art,such as the addition of, e.g., 100-200 μg/mL G418 to the growth medium.

Transfection can be achieved by any of a variety of means known to thoseof ordinary skill in the art including, but not limited to, retroviralinfection. In general, a cell culture may be transfected by incubationwith a mixture of conditioned medium collected from the producer cellline for the vector and DMEM/F12 containing N2 supplements. For example,a placental cell culture prepared as described above may be infectedafter, e.g., five days in vitro by incubation for about 20 hours in onevolume of conditioned medium and two volumes of DMEM/F12 containing N2supplements. Transfected cells carrying a selectable marker may then beselected as described above.

Following transfection, cultures are passaged onto a surface thatpermits proliferation, e.g., allows at least 30% of the cells to doublein a 24 hour period. Preferably, the substrate is apolyornithine/laminin substrate, consisting of tissue culture plasticcoated with polyornithine (10 μg/mL) and/or laminin (10 μg/mL), apolylysine/laminin substrate or a surface treated with fibronectin.Cultures are then fed every 3-4 days with growth medium, which may ormay not be supplemented with one or more proliferation-enhancingfactors. Proliferation-enhancing factors may be added to the growthmedium when cultures are less than 50% confluent.

The conditionally-immortalized placental stem cell lines can be passagedusing standard techniques, such as by trypsinization, when 80-95%confluent. Up to approximately the twentieth passage, it is, in someembodiments, beneficial to maintain selection (by, for example, theaddition of G418 for cells containing a neomycin resistance gene). Cellsmay also be frozen in liquid nitrogen for long-term storage.

Clonal cell lines can be isolated from a conditionally-immortalizedhuman placental stem cell line prepared as described above. In general,such clonal cell lines may be isolated using standard techniques, suchas by limit dilution or using cloning rings, and expanded. Clonal celllines may generally be fed and passaged as described above.

Conditionally-immortalized human placental stem cell lines, which may,but need not, be clonal, may generally be induced to differentiate bysuppressing the production and/or activity of the growth-promotingprotein under culture conditions that facilitate differentiation. Forexample, if the gene encoding the growth-promoting protein is under thecontrol of an externally-regulatable promoter, the conditions, e.g.,temperature or composition of medium, may be modified to suppresstranscription of the growth-promoting gene. For thetetracycline-controlled gene expression system discussed above,differentiation can be achieved by the addition of tetracycline tosuppress transcription of the growth-promoting gene. In general, 1 μg/mLtetracycline for 4-5 days is sufficient to initiate differentiation. Topromote further differentiation, additional agents may be included inthe growth medium.

5.6.4 Assays

Placental stem cells can be used in assays to determine the influence ofculture conditions, environmental factors, molecules (e.g.,biomolecules, small inorganic molecules. etc.) and the like on stem cellproliferation, expansion, and/or differentiation, compared to placentalstem cells not exposed to such conditions.

In one embodiment, placental stem cells can be assayed for changes inproliferation, expansion or differentiation upon contact with amolecule. In one embodiment, for example, provided herein is a method ofidentifying a compound that modulates the proliferation of a pluralityof placental stem cells, comprising contacting said plurality of stemcells with said compound under conditions that allow proliferation,wherein if said compound causes a detectable change in proliferation ofsaid plurality of stem cells compared to a plurality of stem cells notcontacted with said compound, said compound is identified as a compoundthat modulates proliferation of placental stem cells. In a specificembodiment, said compound is identified as an inhibitor ofproliferation. In another specific embodiment, said compound isidentified as an enhancer of proliferation.

In another embodiment, compounds can be identified that modulate theexpansion of a plurality of placental stem cells, comprising contactingsaid plurality of stem cells with said compound under conditions thatallow expansion, wherein if said compound causes a detectable change inexpansion of said plurality of stem cells compared to a plurality ofstem cells not contacted with said compound, said compound is identifiedas a compound that modulates expansion of placental stem cells. In aspecific embodiment, said compound is identified as an inhibitor ofexpansion. In another specific embodiment, said compound is identifiedas an enhancer of expansion.

In another embodiment, a compound that modulates the differentiation ofa placental stem cell can be identified by a method comprisingcontacting said stem cells with said compound under conditions thatallow differentiation, wherein if said compound causes a detectablechange in differentiation of said stem cells compared to a stem cell notcontacted with said compound, said compound is identified as a compoundthat modulates proliferation of placental stem cells. In a specificembodiment, said compound is identified as an inhibitor ofdifferentiation. In another specific embodiment, said compound isidentified as an enhancer of differentiation.

5.6.5 Placental Stem Cell Bank

Stem cells from postpartum placentas can be cultured in a number ofdifferent ways to produce a set of lots, e.g., a set ofindividually-administrable doses, of placental stem cells. Such lotscan, for example, be obtained from stem cells from placental perfusateor from enzyme-digested placental tissue. Sets of lots of placental stemcells, obtained from a plurality of placentas, can be arranged in a bankof placental stem cells for, e.g., long-term storage. Generally,adherent stem cells are obtained from an initial culture of placentalmaterial to form a seed culture, which is expanded under controlledconditions to form populations of cells from approximately equivalentnumbers of doublings. Lots are preferably derived from the tissue of asingle placenta, but can be derived from the tissue of a plurality ofplacentas.

In one embodiment, stem cell lots are obtained as follows. Placentaltissue is first disrupted, e.g., by mincing, digested with a suitableenzyme, e.g., collagenase (see Section 5.2.3, above). The placentaltissue preferably comprises, e.g., the entire amnion, entire chorion, orboth, from a single placenta, but can comprise only a part of either theamnion or chorion. The digested tissue is cultured, e.g., for about 1-3weeks, preferably about 2 weeks. After removal of non-adherent cells,high-density colonies that form are collected, e.g., by trypsinization.These cells are collected and resuspended in a convenient volume ofculture medium, and defined as Passage 0 cells.

Passage 0 cells are then used to seed expansion cultures. Expansioncultures can be any arrangement of separate cell culture apparatuses,e.g., a Cell Factory by NUNC™. Cells in the Passage 0 culture can besubdivided to any degree so as to seed expansion cultures with, e.g.,1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴,1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, or 10×10⁴stem cells. Preferably, from about 2×10⁴ to about 3×10⁴ Passage 0 cellsare used to seed each expansion culture. The number of expansioncultures can depend upon the number of Passage 0 cells, and may begreater or fewer in number depending upon the particular placenta(s)from which the stem cells are obtained.

Expansion cultures are grown until the density of cells in culturereaches a certain value, e.g., about 1×10⁵ cells/cm². Cells can eitherbe collected and cryopreserved at this point, or passaged into newexpansion cultures as described above. Cells can be passaged, e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 timesprior to use. A record of the cumulative number of population doublingsis preferably maintained during expansion culture(s). The cells from aPassage 0 culture can be expanded for 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 doublings, orup to 60 doublings. Preferably, however, the number of populationdoublings, prior to dividing the population of cells into individualdoses, is between about 15 and about 30, preferably about 20 doublings.The cells can be culture continuously throughout the expansion process,or can be frozen at one or more points during expansion.

Cells to be used for individual doses can be frozen, e.g., cryopreservedfor later use. Individual doses can comprise, e.g., about 1 million toabout 100 million cells per ml, and can comprise between about 10⁶ andabout 10⁹ cells in total.

In a specific embodiment, of the method, Passage 0 cells are culturedfor approximately 4 doublings, then frozen in a first cell bank. Cellsfrom the first cell bank are frozen and used to seed a second cell bank,the cells of which are expanded for about another eight doublings. Cellsat this stage are collected and frozen and used to seed new expansioncultures that are allowed to proceed for about eight additionaldoublings, bringing the cumulative number of cell doublings to about 20.Cells at the intermediate points in passaging can be frozen in units ofabout 100,000 to about 10 million cells per ml, preferably about 1million cells per ml for use in subsequent expansion culture. Cells atabout 20 doublings can be frozen in individual doses of between about 1million to about 100 million cells per ml for administration or use inmaking a stem cell-containing composition. In one embodiment, the cellsare diluted to about 2 million/ml in 10% HAS, 10% DMSO in Plasmalyte.

In a preferred embodiment, the donor from which the placenta is obtained(e.g., the mother) is tested for at least one pathogen. If the mothertests positive for a tested pathogen, the entire lot from the placentais discarded. Such testing can be performed at any time duringproduction of placental stem cell lots, including before or afterestablishment of Passage 0 cells, or during expansion culture. Pathogensfor which the presence is tested can include, without limitation,hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, humanimmunodeficiency virus (types I and II), cytomegalovirus, herpesvirus,and the like.

5.6.6 Treatment of Diseases

Provided herein are methods of treating an individual having a disease,disorder or condition, wherein the disease, disorder or condition iscaused by, or is associated with, an inappropriate or undesirable immuneresponse, e.g., a disease, disorder or condition that can be treatedbeneficially by immunosuppression, comprising administering to theindividual placental stem cells. In a specific embodiment, the amount isan amount sufficient to detectably suppress an immune response in theindividual. Such an immune response can be, e.g., proliferation of Tcells in an MLR or regression assay performed using T cells from theindividual.

An individual having a disease, disorder or condition associated with orcaused by an inappropriate or undesirable immune response, e.g., anindividual having, or at risk of developing multiple sclerosis; a personhaving, or at risk of developing, an inflammatory bowel disease, e.g.,Crohn's disease or ulcerative colitis; a person having or at risk ofdeveloping graft-versus-host disease; a person having or at risk ofdeveloping scleroderma; a person having or at risk of developingrheumatoid arthritis; a person having or at risk of developing diabetes;a person having or at risk of developing psoriasis; a person having orat risk of developing mycosis fungoides; and the like, can be treatedwith a plurality of placental stem cells, and, optionally, one or moretherapeutic agents, at any time during the progression of the disease.For example, the individual can be treated immediately after diagnosis,or within 1, 2, 3, 4, 5, 6 days of diagnosis, or within 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more weeks, or 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more years after diagnosis. The individualcan be treated once, or multiple times during the clinical course of thedisease. The individual can be treated, as appropriate, during an acuteattack, during remission, or during a chronic degenerative phase.

The placenta stem cells useful in the treatment of such a disease,disorder or condition can be any of the placental stem cells disclosedherein. In a specific embodiment, the placental stem cells express CD200and HLA-G; express CD73, CD105, and CD200; express CD200 and OCT-4;express CD73, CD105 and HLA-G; express CD73 and CD105, and facilitatethe formation of one or more embryoid-like bodies in a population ofplacental cells when said population is cultured under conditions thatallow for the formation of embryoid-like bodies; or express OCT-4, and(c) facilitate the formation of one or more embryoid-like bodies in apopulation of placental cells when said population is cultured underconditions that allow for the formation of embryoid-like bodies; or anycombination of the foregoing. In a specific embodiment, the placentalstem cells are CD10⁺, CD105⁺, CD200⁺, CD34⁻ placental stem cells. Inanother specific embodiment, the placental stem cells are CD117⁻.

In one embodiment, the individual is administered a dose of about 300million placental stem cells. Dosage, however, can vary according to theindividual's physical characteristics, e.g., weight, and can range from1 million to 10 billion placental stem cells per dose, preferablybetween 10 million and 1 billion per dose, or between 100 million and 50million placental stem cells per dose. The administration is preferablyintravenous, but can be by any medically-acceptable route for theadministration of live cells, e.g., parenterally, subcutaneously,intramuscularly, intraperitoneally, intraocularly, and the like. In oneembodiment, the placental stem cells are from a cell bank. In oneembodiment, a dose of placental stem cells, e.g., from amnion,amnion/chorion, chorion or umbilical cord, is contained within a bloodbag or similar bag, suitable for bolus injection or administration bycatheter.

In another embodiment, provided herein is a method of treating anindividual having a disease, disorder or condition, wherein the disease,disorder or condition is caused by, or is associated with, aninappropriate or undesirable immune response, e.g., a disease, disorderor condition that can be treated beneficially by immunosuppression,comprising administering to the individual culture medium that has beenconditioned placental stem cells, in an amount sufficient to detectablysuppress an immune response in the individual. Such an immune responsecan be, e.g., proliferation of T cells in an MLR or regression assayperformed using T cells from the individual.

Placental stem cells or umbilical cord stem cells, or medium conditionedby placental stem cells or umbilical cord stem cells, can beadministered in a single dose, or in multiple doses. Where placentalstem cells are administered in multiple doses, the doses can be part ofa therapeutic regimen designed to relieve one or more acute symptoms ofdisease, disorder or condition, wherein the disease, disorder orcondition is caused by, or is associated with, an inappropriate orundesirable immune response, or can be part of a long-term therapeuticregimen designed to prevent, or lessen the severity, of a chronic courseof such a disease, disorder or condition.

5.6.7 Treatment of Multiple Sclerosis

In another aspect, provided herein is a method of treating an individualhaving multiple sclerosis, or a symptom associated with multiplesclerosis, comprising administering to the individual a plurality ofplacental stem cells, or medium conditioned by placental stem cells, inan amount and for a time sufficient to detectably modulate, e.g.,suppress an immune response in the individual.

Multiple sclerosis (MS) is a chronic, recurrent inflammatory disease ofthe central nervous system. The disease results in injury to the myelinsheaths surrounding CNS and PNS axons, oligodendrocytes, and the nervecells themselves. The disease is mediated by autoreactive T cells,particularly CD4⁺ T cells, that proliferate, cross the blood-brainbarrier, and enter the CNS under the influence of cellular adhesionmolecules and pro-inflammatory cytokines. The symptoms of MS includesensory disturbances in the limbs, optic nerve dysfunction, pyramidaltract dysfunction, bladder dysfunction, bowel dysfunction, sexualdysfunction, ataxia, and diplopia.

Four different types or clinical courses of MS have been identified. Thefirst, relapsing/remitting MS (RRMS) is characterized by self-limitingattacks of neurological dysfunction that manifest acutely, over thecourse of days to weeks, followed by a period of recovery, sometimesincomplete, over several months. The second type, secondary progressiveMS (SPMS), begins as RRMS but changes such that the clinical coursebecomes characterized by a steady deterioration in function unrelated toacute attacks. The third, primary progressive MS (PPMS), ischaracterized by a steady decline in function from onset, with no acuteattacks. The fourth type, progressive/relapsing MS (PRMS), also beginswith a progressive course, with occasional attacks superimposed on theprogressive decline in function.

Persons having MS are generally evaluated using a motor skillsassessment, optionally with an MRI. For example, one motor skillsassessment, the expanded disability status scale, scores gradations inan affected individual's abilities, as follows:

0.0 Normal neurological examination 1.0 No disability, minimal signs inone FS 1.5 No disability, minimal signs in more than one FS 2.0 Minimaldisability in one FS 2.5 Mild disability in one FS or minimal disabilityin two FS 3.0 Moderate disability in one FS, or mild disability in threeor four FS. Fully ambulatory. 3.5 Fully ambulatory but with moderatedisability in one FS and more than minimal disability in several others4.0 Fully ambulatory without aid, self-sufficient, up and about some 12hours a day despite relatively severe disability; able to walk withoutaid or rest some 500 meters 4.5 Fully ambulatory without aid, up andabout much of the day, able to work a full day, may otherwise have somelimitation of full activity or require minimal assistance; characterizedby relatively severe disability; able to walk without aid or rest some300 meters. 5.0 Ambulatory without aid or rest for about 200 meters;disability severe enough to impair full daily activities (work a fullday without special provisions) 5.5 Ambulatory without aid or rest forabout 100 meters; disability severe enough to preclude full dailyactivities 6.0 Intermittent or unilateral constant assistance (cane,crutch, brace) required to walk about 100 meters with or without resting6.5 Constant bilateral assistance (canes, crutches, braces) required towalk about 20 meters without resting 7.0 Unable to walk beyondapproximately five meters even with aid, essentially restricted towheelchair; wheels self in standard wheelchair and transfers alone; upand about in wheelchair some 12 hours a day 7.5 Unable to take more thana few steps; restricted to wheelchair; may need aid in transfer; wheelsself but cannot carry on in standard wheelchair a full day; May requiremotorized wheelchair 8.0 Essentially restricted to bed or chair orperambulated in wheelchair, but may be out of bed itself much of theday; retains many self-care functions; generally has effective use ofarms 8.5 Essentially restricted to bed much of day; has some effectiveuse of arms retains some self care functions 9.0 Confined to bed; canstill communicate and eat. 9.5 Totally helpless bed patient; unable tocommunicate effectively or eat/swallow 10.0 Death due to MS

In the above scoring system, “FS” refers to the eight functional systemsmeasured, including pyramidal, cerebellar, brainstem, sensory, bowel andbladder, visual, cerebral, and other systems.

Other, similar scoring systems are known, including the Scrippsneurological rating scale, the ambulatory index, and the multiplesclerosis functional composite score (MSFC).

The progress of MS has also been assessed by a determination of theattack rate.

The progress of MS has also been assessed by magnetic resonance imaging,which can detect neural lesions associated with MS (e.g., new lesions,enhancing lesions, or combined unique active lesions).

Thus, in one embodiment, provided herein is a method of treating anindividual having MS, e.g., and individual who has been diagnosed withMS, comprising administering to the individual a plurality of placentalstem cells sufficient to detectably suppress an immune response in theindividual. In a specific embodiment, the MS is relapsing/remitting MS.In another specific embodiment, the MS is secondary progressive MS. Inanother specific embodiment, the MS is primary progressive MS. Inanother specific embodiment, the MS is progressive/relapsing MS. Inanother specific embodiment, the administering detectably improves oneor more symptoms of MS in the individual. In more specific embodiments,the symptom is, e.g., one or more of a sensory disturbance in the limbs,an optic nerve dysfunction, a pyramidal tract dysfunction, a bladderdysfunction, a bowel dysfunction, a sexual dysfunction, ataxia, ordiplopia. In another specific embodiment, said administering results inan improvement on the EDSS scale of at least one half point. In anotherspecific embodiment, said administering results in the maintenance offunction, according to at least one MS scoring system, over the courseof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, In anotherspecific embodiment, said administering results in an improvement on theEDSS scale of at least one point. In another specific embodiment, saidadministering results in an improvement on the EDSS scale of at leasttwo points. In other specific embodiments, said administering results ina detectable improvement on a multiple sclerosis assessment scale or onan MRI. The individual can be treated, as appropriate, during an acuteattack, during remission, or during a chronic degenerative phase. Inanother embodiment, the placental stem cells are administered to afemale having MS, post-partum, to maintain the state of remission orreduced occurrence of relapse experienced during pregnancy.

Also provided herein are methods for the treatment of an individualhaving MS, e.g., an individual who has been diagnosed as having MS,comprising administering to the individual a plurality of placental stemcells sufficient to detectably suppress an immune response in theindividual, wherein the administering detectably improves one or moresymptoms of MS in the individual, and one or more therapeutic agents. Inone embodiment, the therapeutic agent is a glucocorticoid. In specificembodiments, the glucocorticoid is adrenocorticotropic hormone (ACTH),methylprednisolone, or dexamethasone. In another embodiment, thetherapeutic agent is an immunomodulatory or immunosuppressive agent. Invarious specific embodiments, the immunomodulatory or immunosuppressiveagent is IFNβ-1a, IFN-1b, glatiramer acetate, cyclophosphamide,methotrexate, azathioprine, cladribine, cyclosporine or mitoxantrone. Inother embodiments, the therapeutic agent is intravenous immunoglobulin,plasma exchange, or sulfasalazine. In another embodiment, the individualis administered any combination of the foregoing therapeutic agents.

5.6.8 Treatment of Inflammatory Bowel Disease

In one embodiment, placental stem cells, placental stem cellpopulations, and/or compositions comprising placental stem cells orplacental stem cell populations, are used to treat an individual having,or at risk of developing, inflammatory bowel disease (IBD), e.g.,Crohn's disease or ulcerative colitis. Thus, in another aspect, providedherein is a method of treating an individual having inflammatory boweldisease, or a symptom associated with inflammatory bowel disease,comprising administering to the individual a plurality of placental stemcells, or medium conditioned by placental stem cells, in an amount andfor a time sufficient to detectably modulate, e.g., suppress an immuneresponse in the individual.

Crohn's Disease. In one embodiment, the IBD is Crohn's disease,sometimes referred to as ileitis or enteritis. Crohn's disease is achronic disorder that causes inflammation of the digestive tract (alsoreferred to as the gastrointestinal, or GI, tract). Crohn's disease canaffect any part of the GI tract, from mouth to anus, but most commonlyaffects the lower part of the small intestine, referred to as the ileum.Five types of Crohn's disease are known. Gastroduodenal Crohn's diseaseaffects the stomach and duodenum (the highest portion of the smallintestine). Jejunoileitis is Crohn's disease of the jejunum, the longestportion of the small intestine. Ileitis is Crohn's disease of the ileum,the lower portion of the small intestine. Ileocolitis, the most commonform of Crohn's disease, affects the ileum and colon. Finally, Crohn'scolitis (Granulomatous colitis) affects the colon, and is distinguishedfrom ulcerative colitis in that in Crohn's colitis, there are oftenareas of healthy tissue between areas of diseased tissue, and Crohn'scolitis can involve only the colon, without involving the rectum.Crohn's disease is thought to arise from inappropriate reaction of thebody's immune system to antigens in the GI tract, including, e.g., food,beneficial bacteria, etc., resulting in an accumulation of white bloodcells in the lining of the intestines. Inflammation associated withCrohn's disease has also been attributed to the action of the cytokinetumor necrosis factor (TNF-α).

Ulcerative colitis. In another embodiment, the IBD is ulcerativecolitis. Ulcerative colitis is a disease that causes inflammation andsores (ulcers) in the lining of the rectum and/or colon. Ulcers formwhere inflammation has killed the cells that usually line the colon; theulcers typically subsequently bleed and produce pus. When inflammationoccurs in the rectum and lower part of the colon, the disease isreferred to as ulcerative proctitis. If the entire colon is affected,the disease is called pancolitis. If only the left side of the colon isaffected, the disease is referred to as limited or distal colitis.Symptoms of ulcerative colitis include, but are not limited to,abdominal pain, bloody diarrhea, fevers, nausea, abdominal cramps,anemia, fatigue, weight loss, loss of appetite, rectal bleeding, loss ofbodily fluids and nutrients, skin lesions, joint pain, and growthfailure (in children). Ulcerative colitis can also cause complicationssuch as inflammation of the eye, liver disease, and osteoporosis.

Thus, in one embodiment, provided herein is a method of treating anindividual having an inflammatory bowel disease, comprisingadministering a therapeutically effective amount of placental stem cellsto said individual, wherein said therapeutically effective amount is anamount that results in a detectable improvement in at least one symptomof said inflammatory bowel disease (IBD). In a specific embodiment, theIBD is Crohn's disease. In a more specific embodiment, said Crohn'sdisease is gastroduodenal Crohn's disease, jejunoileitis, ileitis,ileocolitis, or Crohn's colitis. In another more specific embodiment,said symptom is a symptom of Crohn's disease. In a more specificembodiment, said symptom of Crohn's disease is inflammation and swellingof a part of the GI tract, abdominal pain, frequent emptying of thebowel, and/or diarrhea. In another more specific embodiment, saidsymptom of Crohn's disease is rectal bleeding, anemia, weight loss,arthritis, skin problems, fever, thickening of the intestinal wall,formation of scar tissue in the intestines, formation of sores or ulcersin the intestine, development of one or more fistulas in the intestinalwall, development of one or more fissures in the anus, development ofnutritional deficiencies (e.g., deficiencies in one or more of proteins,calories, vitamins), development of kidney stones, development ofgallstones, or diseases of the liver or biliary system.

In another more specific embodiment, the IBD is ulcerative colitis. In amore specific embodiment, said ulcerative colitis is ulcerativeproctitis, pancolitis, limited colitis or distal colitis. In anothermore specific embodiment, said symptom is a symptom of ulcerativecolitis. In a more specific embodiment, said symptom is abdominal pain,bloody diarrhea, fevers, nausea, abdominal cramps, anemia, fatigue,weight loss, loss of appetite, rectal bleeding, loss of bodily fluidsand nutrients, skin lesions, joint pain, and growth failure. In anothermore specific embodiment, the symptom is osteoporosis, eye inflammation,or liver disease.

In another specific embodiment, said individual to whom placental stemcells are administered is additionally administered one or more of asecond therapy, wherein said second therapy comprises ananti-inflammatory agent, steroid, immune suppressor, and/or anantibiotic. Examples of anti-inflammatory drugs useful in the treatmentof Crohn's disease or ulcerative colitis include, but are not limitedto, mesalamine, 5-ASA (5-aminosalicylic acid) agents (e.g., ASACOL®(mesalamine, delayed-release), DIPENTUM (Osalazine), PENTASA®(mesalamine controlled-release)), sulfasalazine (a combination of 5-ASAand sulfapyridine), anti-inflammatory antibodies (e.g., Infliximab(REMICADE®)), and the like. Examples of steroids useful in the treatmentof Crohn's disease or ulcerative colitis include, but are not limitedto, cortisone, hydrocortisone, prednisone, methylprednisone, and thelike. Typically, as practiced in the art, the dosage of steroid is firstdelivered in a relatively large dose, followed by smaller dosages asinflammation subsides. Examples of immune suppressors useful in thetreatment of Crohn's disease include, but are not limited to,cyclosporine A, 6-mercaptopurine or azathioprine. Any antibiotic can beused in the treatment of Crohn's disease, including, e.g., ampicillin,sulfonamide, cephalosporin, tetracycline, and/or metronidazole. Inanother specific embodiment, the second therapy is an administration ofporcine whipworms, e.g., ova of Trichuris suis.

5.6.9 Treatment of Graft Versus Host Disease

In another embodiment, provided herein is a method of treating anindividual, e.g., a transplant recipient or individual who will receivea transplant, that has, or is experiencing a symptom of, or is at riskfor developing, graft-versus-host disease (GVHD), comprisingadministering to the individual a therapeutically effective amount ofplacental stem cells, or culture medium conditioned by placental stemcells, wherein the therapeutically effective amount is an amountsufficient to cause a detectable improvement in one or more symptoms ofGVHD, or sufficient to detectably reduce the onset of one or moresymptoms of GVHD

GVHD typically develops after, or as the result of, fully- orpartially-allogeneic tissue transplantation, particularly afterallogeneic hematopoietic stem cell transplantation, and can include oneor more of dermatitis, enteritis and hepatitis that develops typicallywithin 5-100 days of transplantation. GVHD can be acute or chronic.Acute GVHD may be characterized by the appearance of a pruritic orpainful rash, typically by day 5 to 47 after transplantation. HyperacuteGVHD may also be accompanied by fever, generalized erythroderma, anddesquamation. The liver may also become involved, as evidenced by raised(e.g., higher than normal) levels of bilirubin, alanine aminotransferase(ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AP).Acute GVHD can also involve the colon, resulting in diarrhea, internalbleeding, cramping, abdominal pain, and ileus. Chronic GVHD can occur intransplant patients who have experienced acute GVHD, or who werepreviously asymptomatic. Manifestations of chronic GVHD include aburning sensation in the eye, eye irritation, photophobia, and eye paindue to decreased tear secretion; dryness of the mouth, sensitivity tospicy or acidic foods, abdominal pain, dysphagia (difficulty inswallowing), odynophagia (pain on swallowing), weight loss, obstructivelung disease, muscular weakness, neuropathic pain, and/or muscle cramps.

Therefore, in specific embodiments of the method, the therapeuticallyeffective amount of placental stem cells is an amount sufficient tocause a detectable improvement in one or more symptoms of acute GVHD, orsufficient to detectably reduce the onset of one or more symptoms ofacute GVHD. In more specific embodiments, said one or more symptomscomprise dermatitis, pruritic skin, rash, enteritis, hepatitis, fever,erythroderma, desquamation, a raised level of ALT, a raised level ofAST, a raised level of AP; a raised level of Bilirubin; abdominal pain,cramping, internal bleeding, or ileus. In another specific embodiment ofthe method, the therapeutically effective amount of placental stem cellsis an amount sufficient to cause a detectable improvement in one or moresymptoms of chronic GVHD, or sufficient to detectably reduce the onsetof one or more symptoms of chronic GVHD, where said one or more symptomscomprise a burning sensation in the eye, eye irritation, decreased tearproduction, photophobia, eye pain due to decreased tear secretiondryness of the mouth, sensitivity to spicy or acidic foods, abdominalpain, dysphagia (difficulty in swallowing), odynophagia (pain onswallowing), weight loss, obstructive lung disease (including any ofwheezing dyspnea and/or chronic coughing), muscular weakness,neuropathic pain, and/or muscle cramps. In other specific embodiments ofthe method, symptoms of acute GVHD and/or chronic GVHD comprisehyperbilirubinemia, jaundice, portal hypertension, cirrhosis,hemorrhagic conjunctivitis, psudomembrane formation, lagophthalmos,chronic keratoconjunctivitis, sicca, punctuate keratopathy, atrophy ofthe oral mucosa, erythema, development of lichenoid lesions of thebuccal or labial mucosae, bronchiolitis obliterans, vaginitis, vaginalstrictures, autoimmune thrombocytopenia, and/or anemia.

The method is not limited by the nature of the donor or recipient.Transplantation can cross species lines. In preferred embodiments, thedonor and recipient are the same species, e.g., are both human. Thetransplant recipient can be fully- or partially-allogeneic to the donor.The transplantation can be autologous. Transplant recipients or donorscan be less than five years of age, from 1 to 10 years of age, from 5 to15 years of age, from 10 to 20 years of age, from 15 to 25 years of age,from 20 to 30 years of age, from 25 to 35 years of age, from 30 to 40years of age, from 35 to 45 years of age, from 40 to 50 years of age,from 45 to 55 years of age, from 50 to 60 years of age, from 55 to 65years of age, from 60 to 70 years of age, or 70 years of age or older.

GVHD is generally graded by severity of symptoms. For example, in oneembodiment, symptoms of GVHD are staged, and GVHD is graded from 0 (noGVHD)—IV (life-threatening GVHD) according to skin, liver, and/orintestinal symptoms, as shown in Tables 1 and 2:

TABLE 1 Staging of Acute Graft-Versus-Host Disease Liver (BilirubinStage Skin Level, mg/dL) Intestine + Maculopapular 2-3 Diarrhea rash on<25% 500-1000 mL/d of body surface or persistent nausea ++ Maculopapular3-6 Diarrhea rash on 25-50% 1000-1500 of body surface mL/d +++Generalized  6-15 Diarrhea >1500 erythroderma mL/d ++++ Desquamation >15Pain with or and bullae without ileus

TABLE 2 Grading of Acute GVHD Stage Functional Overall Grade Skin LiverGut Impairment 0 (None) 0 0 0 0 I (Mild) + to ++  0 0 0 II (Moderate) +to +++ + + + III (Severe) ++ to +++  ++ to +++  ++ to +++  ++ IV (Life++ to ++++ ++ to ++++ ++ to ++++ +++ threatening)

Thus, in another embodiment of the method, the therapeutically effectiveamount of placental stem cells is an amount sufficient to cause animprovement in one or more symptoms of graft-versus-host disease, e.g.,in an individual, e.g., an individual that has received a transplant(transplant recipient), such that said graft-versus-host disease isreduced in grade by at least one step. In specific embodiments, saidgraft-versus-host disease is reduced from grade IV to grade III; fromgrade IV to grade II; from grade IV to grade I; from grade IV to grade0; from grade III to grade II; from grade III to grade I; from grade IIIto grade 0; from grade II to grade I; from grade II to grade 0; or fromgrade I to grade 0. In another embodiment of the method, thetherapeutically effective amount of placental stem cells is an amountsuch that graft-versus-host disease in said individual does not developpast grade 0, grade I, grade II or grade II within 10, 20, 30, 40, 50,60, 70, 80, 90, or 100 days post-transplantation.

In various specific embodiments, the individual having, or who is atrisk for developing, GVHD are individuals receiving allogeneichematopoietic cell transplants (e.g., individuals receiving no GVHDprophylaxis; older individuals; recipients of HLA-nonidenticalhematopoietic stem cells; recipients of grafts from allosensitizeddonors; recipients of grafts from unrelated donors); individualsreceiving solid organ transplants, particularly transplants of organscomprising lymphoid tissue, e.g., small bowel transplants; andindividuals receiving unirradiated blood products (e.g., neonates andfetuses, individuals having congenital immunodeficiency syndromes,individuals receiving immunosuppressive chemotherapy, individualsreceiving directed blood donations from partially HLA-identical,HLA-homologous donors), individuals receiving composite tissueallografts (that is, allografts having more than one tissue type); andthe like. GVHD can also occur after autologous or syngeneichematopoietic cell transplantation. In another specific embodiment, theindividual has received radiation (e.g., has been irradiated) at asub-lethal or lethal dose as an adjunct to transplantation.

In specific embodiments, the placental or umbilical cord stem cells areadministered to the individual within 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 day prior to transplantation. In another specificembodiment, the placental or umbilical cord stem cells are administeredconcurrently with transplantation. In another specific embodiment, theplacental or umbilical cord stem cells are administered within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days of transplantation.Administration of the placental or umbilical cord stem cells can beperformed multiple times, e.g., multiple times before, with or aftertransplantation, or any combination thereof. In another embodiment,umbilical cord or placental stem cells are administered at any timepost-transplantation when graft-versus-host disease of Grade II or worseis manifested in the individual (transplant recipient).

In another embodiment of the method, the individual, e.g., transplantrecipient or an individual who will receive a transplant, isadministered placental stem cells or umbilical cord stem cells andadditionally at least one other therapeutic agent. In a specificembodiment, the therapeutic agent is athymocyte globulin, mycophenolatemofetil, sirolimus, Campath-1H, keratinocyte growth factor (KGF),suberoylanilide hydroxamic acid (SAHA), cortisone, hydrocortisone,prednisone, or methylprednisone. In another specific embodiment, thetherapeutic agent is an immunosuppressive agent or immunomodulatoryagent. Immunosuppressive agents and immunomodulatory agents applicableto GVHD are known in the art and include, but are not limited to,methothrexate, leflunomide, cyclophosphamide, cyclosporine A, macrolideantibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP),corticosteroids, steroids, mycophenolate mofetil, rapamycin (sirolimus),mizoribine, deoxyspergualin, brequinar, malononitriloamindes (e.g.,leflunamide), T cell receptor modulators, and cytokine receptormodulators. peptide mimetics, and antibodies (e.g., human, humanized,chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)₂ fragments orepitope binding fragments), nucleic acid molecules (e.g., antisensenucleic acid molecules and triple helices), small molecules, organiccompounds, and inorganic compounds. In particular, immunomodulatoryagents include, but are not limited to, methothrexate, leflunomide,cyclophosphamide, cytoxan, Immuran, cyclosporine A, minocycline,azathioprine, antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone(MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin(sirolimus), mizoribine, deoxyspergualin, brequinar,malononitriloamindes (e.g., leflunamide), T cell receptor modulators,and cytokine receptor modulators. Examples of T cell receptor modulatorsinclude, but are not limited to, anti-T cell receptor antibodies (e.g.,anti-CD4 antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9. Is (IDEC andSKB), mAB 4162W94, ORTHOCLONE® and OKTcdr4a (Janssen-Cilag)), anti-CD3antibodies (e.g., NUVION® (Product Design Labs), OKT3 (Johnson &Johnson), or Rituxan (IDEC)), anti-CD5 antibodies (e.g., an anti-CD5ricin-linked immunoconjugate), anti-CD7 antibodies (e.g., CHH-380(Novartis)), anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies(e.g., IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH® 1H(Ilex)), anti-CD2 antibodies, anti-CD1a antibodies (e.g., Xanelim(Genentech)), and anti-B7 antibodies (e.g., IDEC-114) (IDEC))),CTLA4-immunoglobulin, thalidomide, or one of the compounds in Section5.6.6, above. In a specific embodiment, a T cell receptor modulator is aCD2 antagonist. In other embodiments, a T cell receptor modulator is nota CD2 antagonist. In another specific embodiment, the agent is antibodyMEDI-501 (T10B9). In another specific embodiment, a T cell receptormodulator is a CD2 binding molecule, preferably MEDI-507. In otherembodiments, a T cell receptor modulator is not a CD2 binding molecule.Any combination of the above therapeutic agents, suitable for treatmentof GVHD or a symptom of GVHD, can be administered. Such therapeuticagents can be administered in any combination with the placental stemcells or umbilical cord stem cells, at the same time or as a separatecourse of treatment.

5.6.10 Treatment of Rheumatoid Arthritis

In another embodiment, provided herein is a method of treating anindividual that has, or is experiencing a symptom of, or is at risk fordeveloping, rheumatoid arthritis (RA), comprising administering to theindividual a therapeutically effective amount of placental stem cells orumbilical cord stem cells, or culture medium conditioned by placentalstem cells or umbilical cord stem cells, wherein the therapeuticallyeffective amount is an amount sufficient to cause a detectableimprovement in one or more symptoms of RA, or sufficient to detectablyreduce the onset of one or more symptoms of RA. Rheumatoid arthritis isa chronic, inflammatory autoimmune condition in which the body's immunesystem attacks the joints, and, typically, other tissues of the body.

In a specific embodiment, the administration is sufficient to cause adetectable improvement in one or more symptoms of RA, or sufficient todetectably reduce the onset of one or more symptoms of RA, in at leastone joint in the individual with RA. In another specific embodiment, theadministration is sufficient to cause a detectable improvement in one ormore symptoms of RA, or sufficient to detectably reduce the onset of oneor more symptoms of RA, in at least one non-joint tissue in theindividual with RA. Examples of non-joint tissue that can be affected byRA include, but are not limited to, skin (dermis), lungs, autoimmunesystem or blood, renal tissue, cardiovascular tissue, ocular tissue, orneurological tissue.

In specific embodiments, the symptom of RA is, without limitation,morning stiffness (e.g., over an hour in duration), soft-tissue swellingof one or more joints or joint groups, joint pain, subcutaneous nodules,rheumatoid factor present at above 95^(th) percentile, or radiologicalchanges suggestive of joint erosion.

In a specific embodiment, the administration is sufficient to cause adetectable improvement in one or more conditions adjunct to RA, orsufficient to detectably reduce the onset of one or more conditionsadjunct to RA. Examples of such conditions include, but are not limitedto, pyoderma gangrenosum, neutrophilic dermatosis, Sweet's syndrome,viral infection, erythema nodosum, lobular panniculitis, atrophy ofdigital skin, palmar erythema, diffuse thinning (rice paper skin), skinfragility, subcutaneous nodules on an exterior surface, e.g., on theelbows, fibrosis of the lungs (e.g., as a consequence of methotrexatetherapy), Caplan's nodules, vasculitic disorders, nail fold infarcts,neuropathy, nephropathy, amyloidosis, muscular pseudohypertrophy,endoscarditis, left ventricular failure, valulitis, scleromalacia,mononeuritis multiplex, atlanto-axial subluxation, and the like.

In another embodiment of the method, the individual having RA isadministered placental stem cells or umbilical cord stem cells andadditionally at least one other therapeutic agent. In specificembodiments, the therapeutic agent is, e.g., an analgesic, or ananti-inflammatory agent. In another specific embodiment, the therapeuticagent is a disease-modifying antirheumatic drug (DMARD). In a morespecific embodiment, the DMARD is one or more of a xenobiotic (e.g.,azathioprine, cyclosporine A, D-penicillamine, gold salts,hydroxychloroquine, leflunomide, methotrexate, minocycline orsulfasalazine) or a biological agent (e.g., tumor necrosis factor alpha(TNF-α) blockers, such as etanercept (ENBREL®), infliximab (REMICADE®),adalimumab (HUMIRA®), or one of the compounds disclosed in Section5.6.8, above; interleukin-1 blockers; anti-B cell (CD20) antibody (e.g.,rituximab or RITUXAN®); or blockers of T cell activation (e.g.,abatacept or ORENCIA®). In another more specific embodiment, theanalgesic or anti-inflammatory agent is a glucocorticoid, anon-steroidal anti-inflammatory drug, acetaminophen, ibuprofen, aspirin,an opiate, or lidocaine (topical). Any combination of the abovetherapeutic agents, suitable for treatment of GVHD or a symptom of GVHD,can be administered. Such therapeutic agents can be administered in anycombination with the placental stem cells or umbilical cord stem cells,at the same time or as a separate course of treatment.

In a specific embodiment, a plurality of the placental stem cells orumbilical cord stem cells administered to an individual having RA havebeen genetically engineered to express a polypeptide therapeutic for RA.In a more specific embodiment, the polypeptide therapeutic for RA isIL-1Ra (interleukin-1 receptor antagonist). In another more specificembodiment, the polypeptide therapeutic for RA is a fusion proteincomprising IL-1Ra and DHFR (dihydrofolate reductase). In a more specificembodiment, the placental stem cell or umbilical cord stem cell istransformed with a nucleic acid encoding IL-1Ra-DHFR fusion protein,wherein expression of the fusion protein is enhanced by an antifolate,e.g., methotrexate. In another specific embodiment, a plurality of asecond type of stem cell is administered to the individual having RA,wherein a plurality of the second type of stem cell has been geneticallyengineered to express a polypeptide therapeutic for RA, e.g., any of thepolypeptides disclosed above. In an even more specific embodiment, thenucleic acid encodes IL-1Ra-DHFR-IRES-Luc, where IRES is an internalribosomal entry site, and Luc is luciferase. In another specificembodiment, said nucleic acid comprises a nucleotide sequence thatenables control of expression of the IL-1Ra or IL-1Ra-DHFR fusionpolypeptide.

Genetically engineered placental stem cells, umbilical cord stem cells,or other kind of stem cell, used to treat RA, can be administered to anindividual with RA in any combination with such stem cells that have notbeen genetically modified.

5.6.11 Treatment of Scleroderma

In another embodiment, provided herein is a method of treating anindividual that has, or is experiencing a symptom of, or is at risk fordeveloping, scleroderma, comprising administering to the individual atherapeutically effective amount of placental stem cells or umbilicalcord stem cells, or culture medium conditioned by placental stem cellsor umbilical cord stem cells, wherein the therapeutically effectiveamount is an amount sufficient to cause a detectable improvement in oneor more symptoms of scleroderma, or sufficient to detectably reduce theonset of one or more symptoms of scleroderma.

Scleroderma is a chronic disease characterized by excessive deposits ofcollagen in the skin or other organs. Scleroderma can be localized orgeneralized. The localized form of the disease, while disabling, tendsnot to be fatal. The generalized form of the disease, manifesting asdiffuse scleroderma or systemic sclerosis, can be fatal as a result ofheart, kidney, lung or intestinal damage. The three types of sclerodermaare diffuse scleroderma and limited (CREST syndrome) scleroderma, whichare systemic, and morphea/linear scleroderma, which is limited to theskin. Diffuse scleroderma is the most severe form, with victimsexperiencing rapid onset, widespread skin hardening, and significantinternal organ damage, particularly to the lungs and gastrointestinaltract.

The limited form of scleroderma is much milder, exhibiting a sloweronset and progression. Skin hardening is usually confined to the handsand face, internal organ involvement is less severe than in the diffuseform. Typically, Raynaud's phenomenon may precede scleroderma by severalyears. Raynaud's phenomenon is due to vasoconstriction of the smallarteries of exposed peripheries—particularly the hands and feet—in thecold, and is classically characterized by a triphasic color change—firstwhite, then blue and finally red on rewarming. The limited form is oftenreferred to as CREST syndrome, where “CREST” is an acronym for the fivemain features, calcinosis (calcium deposits in soft tissue, e.g., theskin), Raynaud's syndrome, esophageal dysmotility, sclerodactyly(scleroderma of the fingers), and telangiectasia (spider veins).

Development of scleroderma has been correlated with the presence ofautoantibodies, particularly anti-centromere andanti-scl70/anti-topoisomerase antibodies. Up to 90% of affectedindividuals have a detectable anti-nuclear antibody. Anti-centromereantibody is more common in the limited form (80-90%) than in thesystemic form (10%), and anti-scl70 is more common in the diffuse form(30-40%) and in African-American patients.

Thus, in the method of treatment provided herein, the administration ofplacental stem cells or umbilical cord stem cells inhibits thedevelopment of, reduces the severity of, or reduces the progression of,one or more symptoms of scleroderma. In one embodiment, the sclerodermais limited scleroderma. In another embodiment, the scleroderma isdiffuse scleroderma. In another embodiment, the scleroderma is morphea.In another specific embodiment, the symptom is one or more of hardeningof the skin of the face, hardening of the skin of the fingers, Reynaud'ssyndrome, inappropriate vasoconstriction in an extremity, calcinosis,telangiectasia, or esophageal dysmotility. In another specificembodiment, administration of placental stem cells or umbilical cordstem cells detectably reduces the amount or concentration in amilliliter of blood from the individual of one or more anti-nuclearantibodies, e.g., an anti-centromere antibody or an anti-topoisomeraseantibody.

In another embodiment, the method of treatment provided herein comprisesthe administration of a second therapy or therapeutic agent, wherein thesecond therapy or therapeutic agent is an anti-inflammatory drug, e.g.,a steroidal anti-inflammatory drug, or a non-steroidal anti-inflammatorydrug (NSAID), acetaminophen, naproxen, ibuprofen, acetylsalicylic acid,and the like. In a more specific embodiment in which an NSAID isadministered, a proton pump inhibitor (PPI), e.g., omeprazole is alsoadministered. In another embodiment, the second therapy is animmunosuppressant compound such as mycophenolate mofetil,cyclophosphamide or methotrexate. In another embodiment, where theaffected individual has digital ulcerations and pulmonary hypertension,a vasodilator such as prostacyclin (iloprost) is administered.

In another embodiment, the second therapy is a second type of cell,e.g., hematopoietic stem cells, e.g., CD34⁺ hematopoietic stem cells, inone or more doses of from about 10⁵ cells/kg to about 10⁹ cells/kg. In aspecific embodiment, said second type of stem cell is a mesenchymal stemcell, e.g., a bone marrow-derived mesenchymal stem cell. The second typeof stem cell, e.g., hematopoietic stem cell or mesenchymal stem cell,can be administered with the placental stem cells in any ratio, e.g.,about 100:1, 75:1, 50:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10,1:15, 1:20, 1:25, 1:50, 1:75 or 1:100. Such mesenchymal stem cells canbe obtained commercially or from an original source, e.g., bone marrow,bone marrow aspirate, adipose tissue, and the like.

Any combination of the above therapeutic agents, suitable for treatmentof scleroderma or a symptom of scleroderma, can be administered. Suchtherapeutic agents can be administered in any combination with theplacental stem cells or umbilical cord stem cells, at the same time oras a separate course of treatment.

Placental stem cells or umbilical cord stem cells can be administered tothe individual suffering scleroderma in the form of a pharmaceuticalcomposition, e.g., a pharmaceutical composition suitable for, e.g.,intravenous, intramuscular or intraperitoneal injection.

5.6.12 Treatment of Psoriasis

In another embodiment, provided herein is a method of treating anindividual that has, or is experiencing a symptom of, or is at risk fordeveloping, psoriasis, comprising administering to the individual atherapeutically effective amount of placental stem cells or umbilicalcord stem cells, or culture medium conditioned by placental stem cellsor umbilical cord stem cells, wherein the therapeutically effectiveamount is an amount sufficient to cause a detectable improvement in oneor more symptoms of psoriasis, sufficient to detectably reduce the onsetof one or more symptoms of psoriasis, or sufficient to reduce theprogress of psoriasis.

Psoriasis is a disease affecting the skin and joints, which commonlycauses red scaly patches, called psoriatic plaques, to appear on theskin. Psoriatic patches are areas of inflammation and excessive skinproduction. Skin rapidly accumulates at these sites and takes asilvery-white appearance. Plaques frequently occur on the skin of theelbows and knees, but can affect any area including the scalp andgenitals. Psoriasis is hypothesized to be immune-mediated.

Several different types of psoriasis have been identified, as follows:

Plaque psoriasis (psoriasis vulgaris), the most common form ofpsoriasis, typically appears as raised areas of inflamed skin coveredwith silvery white scaly skin, called plaques.

Flexural psoriasis (inverse psoriasis) appears as smooth inflamedpatches of skin occurring in skin folds, for example, around thegenitals (between the thigh and groin), the armpits, under an overweightstomach, and under the breasts.

Guttate psoriasis manifests as numerous small oval (teardrop-shaped)spots that appear over large areas of the body, such as the trunk,limbs, and scalp. Guttate psoriasis is associated with streptococcalthroat infection.

Pustular psoriasis appears as raised bumps that are filled withnon-infectious pus (pustules). Pustular psoriasis can be localized,commonly to the hands and feet (palmoplantar pustulosis), or generalizedwith widespread patches occurring randomly on any part of the body.

Nail psoriasis produces a variety of changes in the appearance of fingerand toe nails, including discoloration under the nail plate, pitting ofthe nails, lines going across the nails, thickening of the skin underthe nail, and the loosening (onycholysis) and crumbling of the nail.

Psoriatic arthritis involves joint and connective tissue inflammation,e.g., in the joints of the fingers and toes, which can result in asausage-shaped swelling of the fingers and toes known as dactylitis.Psoriatic arthritis can also affect the hips, knees and spine(spondylitis).

Erythrodermic psoriasis manifests as the widespread inflammation andexfoliation of the skin over most of the body surface. It may beaccompanied by severe itching, swelling and pain. It is often the resultof an exacerbation of unstable plaque psoriasis, particularly followingthe abrupt withdrawal of systemic treatment. This form of psoriasis canbe fatal, as the extreme inflammation and exfoliation disrupt the body'sability to regulate temperature and for the skin to perform barrierfunctions.

In one embodiment, the invention provides for the administration of aneffective dose placental stem cells to an individual affected withpsoriasis, wherein said effective dose is an amount of placental stemcells sufficient, e.g., to cause a detectable improvement in, reduce theseverity of, or reduce the progression of, one or more of the symptomsof psoriasis listed above.

The severity of psoriasis can be evaluated, e.g., by the Psoriasis AreaSeverity Index (PAST). PASI combines the assessment of the severity oflesions and the area affected into a single score in the range 0 (nodisease) to 72 (maximal disease).

To calculate the PASI, the body is divided into four sections: legs, thebody (trunk area (stomach, chest, back, etc.); arms; and head. Each ofthese areas is scored by itself, and then the four scores are combinedinto the final PASI. For each section, the percent of area of skinafflicted with psoriasis is estimated and then transformed into a gradefrom 0 to 6, as follows:

Percent Area Involved Grade  0 0 <10 1  10-29% 2 30-49 3 50-69 4 70-89 5 90-100 6

The severity is estimated by four different parameters, graded from 0 to4: itching, erythema (redness), scaling and thickness. The sum of allfour severity parameters is then calculated for each section of skin,multiplied by the area score for that area and multiplied by weight ofrespective section (0.1 for head, 0.2 for arms, 0.3 for body and 0.4 forlegs). Example:(I_(body)+E_(body)+S_(body)+T_(body))×A_(body)×0.3=Total_(body). At theend the total PASI is calculated as a sum of PASIs for all four skinsections.

The degree of severity can also be assessed by photographing anindividual afflicted with psoriasis, and calculating, by computer, thepercent body area covered by psoriatic lesions.

Thus, in a specific embodiment of the method of treatment providedherein, the psoriasis is plaque psoriasis (psoriasis vulgaris), flexuralpsoriasis (inverse psoriasis), guttate psoriasis, pustular psoriasis,nail psoriasis, psoriatic arthritis, or erythrodermic psoriasis. In aspecific embodiment of the method, the therapeutically effective amountof placental stem cells or umbilical cord stem cells, or culture mediumconditioned by placental stem cells or umbilical cord stem cells is anamount sufficient to cause an improvement in, a delay in onset of, or alessening of the progression of one or more symptoms of psoriasis, wheresaid one or more symptoms are scaling of the skin, redness of the skin,thickening of the skin, formation of plaques, discoloration under thenail plate, pitting of the nails, lines going across the nails,thickening of the skin under the nail, onycholysis, development ofpustules, joint or connective tissue inflammation, inflammation of theskin, or exfoliation of the skin. In another embodiment, thetherapeutically effective amount of placental stem cells or umbilicalcord stem cells, or culture medium conditioned by placental stem cellsor umbilical cord stem cells is an amount sufficient to cause a 5, 10,15, 20, 25, 30, 35, 40 or more point reduction in the Psoriasis AreaSeverity Index.

In another embodiment, the therapeutically effective amount of placentalstem cells or umbilical cord stem cells, or culture medium conditionedby placental stem cells or umbilical cord stem cells, is administered inconjunction with a second therapy. The second therapy can be topical,e.g., creams or ointments comprising one or more of a corticosteroid(e.g., desoximetasone), a vitamin D₃ analog (e.g., calcipotriol),anthralin, argan oil, a retinoid, or coal tar. In another specificembodiment, the second therapy comprises one or more exposures, e.g.,for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20 minutes,to ultraviolet light, e.g., UVB of a wavelength between about 280 nm toabout 315 nm, particularly about 311 nm to about 312 nm. In anotherspecific embodiment, the second therapy comprises topical administrationof psoralen in combination with exposure to UVA light. In anotherspecific embodiment, the second therapy comprises one or more systemicadministrations of one or more of, e.g., methotrexate, cyclosporine, aretinoid, tioguanine, hydroxyurea, sulfasalazine, mycophenolate mofetil,azathioprine, oral tacrolimus and/or a fumaric acid ester.

5.6.13 Treatment of Lupus Erythematosus

In another embodiment, provided herein is a method of treating anindividual that has, or is experiencing a symptom of, or is at risk fordeveloping, lupus erythematosus (LE), comprising administering to theindividual a therapeutically effective amount of placental stem cells orumbilical cord stem cells, or culture medium conditioned by placentalstem cells or umbilical cord stem cells, wherein the therapeuticallyeffective amount is an amount sufficient to cause a detectableimprovement in one or more symptoms of LE, sufficient to detectablyreduce the onset of one or more symptoms of LE, or sufficient to reducethe progress of LE.

Symptoms of LE are numerous, and the disease may progress differently indifferent individuals. Symptoms can include dermatologicalmanifestations (e.g., malar rash (also called butterfly rash), discoidlupus (thick, red scaly patches on the skin), alopecia, mouth, nasal,and vaginal ulcers, and/or lesions on the skin); musculoskeletalmanifestations (e.g., joint pain); hematological manifestations (e.g.,anemia and iron deficiency, lower than normal platelet and white bloodcell counts, antiphospholipid antibody syndrome (a thrombotic disorderin which autoantibodies to phospholipids are present in the patient'sserum), and/or presence of anticardiolipin antibody in the blood);cardiac manifestations (e.g., pericarditis, myocarditis, and/orendocarditis); pulmonary manifestations (e.g., lung and/or pleurainflammation, pleuritis, pleural effusion, lupus pneumonitis, chronicdiffuse interstitial lung disease, pulmonary hypertension, pulmonaryemboli, and/or pulmonary hemorrhage); hepatic manifestations (e.g.,autoimmune hepatitis; jaundice; presence of antinuclear antibody (ANA),smooth muscle antibody (SMA), liver/kidney microsomal antibody (LKM-1)and/or anti-mitochondrial antibody (AMA) in the bloodstream); renalmanifestations (e.g., painless hematuria or proteinuria, lupusnephritis, renal failure, and/or development of membranousglomerulonephritis with “wire loop” abnormalities); neurologicalmanifestations (e.g., seizures, psychosis, abnormalities in thecerebrospinal fluid); T-cell abnormalities (e.g., deficiency in CD45phosphatase and/or increased expression of CD40 ligand); and/ornonspecific manifestations (e.g., lupus gastroenteritis, lupuspancreatitis, lupus cystitis, autoimmune inner ear disease,parasympathetic dysfunction, retinal vasculitis, systemic vasculitis,increased expression of FcεRIγ, increased and sustained calcium levelsin T cells, increase of inositol triphosphate in the blood, reduction inprotein kinase C phosphorylation, reduction in Ras-MAP kinase signaling,and/or a deficiency in protein kinase A I activity.

Thus, in a specific embodiment, said therapeutically effective amount ofplacental stem cells or umbilical cord stem cells, or culture mediumconditioned by placental stem cells or umbilical cord stem cells is anamount effective to cause a detectable improvement in one or moresymptoms of LE, sufficient to detectably reduce the onset of one or moresymptoms of LE, or sufficient to reduce the worsening of one or moresymptoms of LE, wherein said one or more symptoms comprises one or moredermatological, hematological, musculoskeletal, neurological, renal,hepatic, or T-cell manifestations of LE. In another specific embodiment,said therapeutically effective amount is an amount sufficient to cause adetectable improvement in one or more symptoms of LE, sufficient todetectably reduce the onset of one or more symptoms of LE, or sufficientto reduce the worsening of one or more symptoms of LE, wherein said oneor more symptoms comprise malar rash, butterfly rash, discoid lupus,alopecia, mouth, nasal, and vaginal ulcers, lesions on the skin, jointpain anemia and/or iron deficiency, lower than normal platelet and whiteblood cell counts, antiphospholipid antibody syndrome, presence ofanticardiolipin antibody in the blood, pericarditis, myocarditis,endocarditis, lung and/or pleural inflammation, pleuritis, pleuraleffusion, lupus pneumonitis, chronic diffuse interstitial lung disease,pulmonary hypertension, pulmonary emboli, pulmonary hemorrhage,autoimmune hepatitis; jaundice; presence of antinuclear antibody (ANA),smooth muscle antibody (SMA), liver/kidney microsomal antibody (LKM-1)and/or anti-mitochondrial antibody (AMA) in the bloodstream, painlesshematuria or proteinuria, lupus nephritis, renal failure, and/ordevelopment of membranous glomerulonephritis with “wire loop”abnormalities); neurological manifestations (e.g., seizures, psychosis,abnormalities in the cerebrospinal fluid); T-cell abnormalities (e.g.,deficiency in CD45 phosphatase and/or increased expression of CD40ligand); and/or nonspecific manifestations (e.g., lupus gastroenteritis,lupus pancreatitis, lupus cystitis, autoimmune inner ear disease,parasympathetic dysfunction, retinal vasculitis, systemic vasculitis,increased expression of FcεRIγ, increased and sustained calcium levelsin T cells, increase of inositol triphosphate in the blood, reduction inprotein kinase C phosphorylation, reduction in Ras-MAP kinase signaling,and/or a deficiency in protein kinase A I activity.

Placental stem cells or umbilical cord stem cells can be administered tothe individual suffering scleroderma in the form of a pharmaceuticalcomposition, e.g., a pharmaceutical composition suitable for, e.g.,intravenous, intramuscular or intraperitoneal injection.

5.6.14 Treatment of Mycosis Fungoides

In another embodiment, provided herein is a method of treating anindividual that has, or is experiencing a symptom of, or is at risk fordeveloping, mycosis fungoides, comprising administering to theindividual a therapeutically effective amount of placental stem cells orumbilical cord stem cells, or culture medium conditioned by placentalstem cells or umbilical cord stem cells, wherein the therapeuticallyeffective amount is an amount sufficient to cause a detectableimprovement in one or more symptoms of mycosis fungoides, sufficient todetectably reduce the onset of one or more symptoms of mycosisfungoides, or sufficient to reduce the progress of mycosis fungoides.

Mycosis fungoides is the most common of the cutaneous T-cell lymphomas,a group of rare cancers that grow in the skin. Sezary syndrome, a morerare form in which the T cells affect the peripheral blood as well asthe skin, occurs in about 5% of all cases of mycosis fungoides. Mycosisfungoides generally progresses in stages defined by the skin symptoms:(1) patch phase, in which the skin has flat, red patches, or, indark-skinned individuals very light or very dark patches, that are veryitchy, and may be raised and hard (plaques); (2) skin tumors phase, inwhich red-violet raised lumps (nodules) appear, which may be dome-shaped(like a mushroom) or ulcerated; (3) skin redness (erythroderma) stage,in which the individual's skin develops large red areas that are veryitchy and scaly, and in which skin of the palms and soles may thickenand crack; and (4) lymph node stage, in which mycosis fungoides beginsto move to other parts of the body via the lymph nodes, which becomeinflamed, and often cancerous, and may spread to the liver, lungs, orbone marrow.

Thus in a specific embodiment, the therapeutically effective amount ofplacental stem cells or umbilical cord stem cells, or culture mediumconditioned by placental stem cells or umbilical cord stem cells is anamount effective to cause a detectable improvement in one or moresymptoms of mycosis fungoides, sufficient to detectably reduce the onsetof one or more symptoms of mycosis fungoides, or sufficient to reducethe worsening of one or more symptoms of LE, wherein said one or moresymptoms comprises one or more dermatological, hematological,musculoskeletal, neurological, renal, hepatic, or T-cell manifestationsof mycosis fungoides. In another specific embodiment, saidtherapeutically effective amount is an amount sufficient to cause adetectable improvement in one or more symptoms of mycosis fungoides,sufficient to detectably reduce the onset of one or more symptoms ofmycosis fungoides, or sufficient to reduce the worsening of one or moresymptoms of mycosis fungoides, wherein said one or more symptomscomprise itchy light or dark patches on the skin, skin plaques,development of skin tumors, raised bumps on the skin, development ofskin areas that are red, scaly and itchy, thickening of the skin of thesoles or palms, cracking of the skin of the soles or palms, orinflammation of the lymph nodes.

In another embodiment, the therapeutically effective amount of placentalstem cells or umbilical cord stem cells, or culture medium conditionedby placental stem cells or umbilical cord stem cells, is administered inconjunction with a second therapy or second therapeutic agent. In morespecific embodiments, the second therapy or therapeutic agent is one ormore of exposure of an affected area of said individual to sunlight orultraviolet light, topical steroids, local superficial radiotherapy,total skin electron beam radiation, application of organic (Manuka)honey to skin affected by erythroderma, or biological therapies. In amore specific embodiment, said biological therapies compriseadministration to the individual of one or more of an interferon, aretinoid, a rexinoid, vorinostat (e.g., ZOLINZA®).

5.6.15 Treatment of Diabetes

In another embodiment, provided herein is a method of treating anindividual that has, or is experiencing a symptom of, or is at risk fordeveloping, diabetes, comprising administering to the individual atherapeutically effective amount of adherent placental stem cells oradherent umbilical cord stem cells, or culture medium conditioned byplacental stem cells or umbilical cord stem cells, wherein thetherapeutically effective amount is an amount sufficient to cause adetectable improvement in one or more symptoms of diabetes, sufficientto detectably reduce the onset of one or more symptoms of diabetes, orsufficient to reduce the progress of diabetes. In a specific embodiment,the diabetes is diabetes mellitus Type 1, also known as Type 1 diabetes,Type I diabetes, T1D, or insulin-dependent diabetes mellitus (IDDM).

The placental stem cells can be administered one or more times duringthe course of the disease. Preferably, the stem cells are administeredwithin 1, 2, 3, 4, 5, or 6 days, or 1 week, of first diagnosis. In oneembodiment, said therapeutically effective amount of the stem cells isan amount sufficient to reverse, reduce the severity of, or otherwiseameliorate a symptom of diabetes mellitus Type 1, including abnormallyhigh blood sugar, lack of insulin resistance as determined by a glucosetolerance test, fatigue, or loss of consciousness.

The placental stem cells can be administered in conjunction with asecond therapy, e.g., transplanted pancreatic tissue and/or islet cells;autologous or allogeneic stem cell therapy, and the like.

5.6.16 Second Therapeutic Compositions and Second Therapies

In any of the above methods of treatment, the method can comprise theadministration of a second therapeutic composition or second therapy.The recitation of specific second therapeutic compounds or secondtherapies in the methods of treating specific diseases, above, are notintended to be exclusive. For example, any of the diseases, disorders orconditions discussed herein can be treated with any of theanti-inflammatory compounds or immunosuppressive compounds describedherein. In embodiments in which placental stem cells are administeredwith a second therapeutic agent, or with a second type of stem cell, theplacental stem cells and second therapeutic agent and/or second type ofstem cell can be administered at the same time or different times, e.g.,the administrations can take place within 1, 2, 3, 4, 5, 6, 7, 8, 9 10,20, 30, 40, or 50 minutes of each other, or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 18, 20, or 22 hours of each other, or within 1, 2, 3, 4,5, 6, 7 8, 9 or 10 days of each other.

In a specific embodiment, treatment of a disease, disorder or conditionrelated to or caused by an inappropriate, deleterious or harmful immuneresponse comprises administration of a second type of stem cell, orpopulation of a second type of stem cell. In a specific embodiment, saidsecond type of stem cell is a mesenchymal stem cell, e.g., a bonemarrow-derived mesenchymal stem cell. In other embodiments, the secondtype of stem cell is a multipotent stem cell, a pluripotent stem cell, aprogenitor cell, a hematopoietic stem cell, e.g., a CD34⁺ hematopoieticstem cell, an adult stem cell, an embryonic stem cell or an embryonicgerm cell. The second type of stem cell, e.g., mesenchymal stem cell,can be administered with the placental stem cells or umbilical cord stemcells in any ratio, e.g., a ratio of about 100:1, 75:1, 50:1, 25:1,20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:50, 1:75 or1:100. Mesenchymal stem cells can be obtained commercially or from anoriginal source, e.g., bone marrow, bone marrow aspirate, adiposetissue, and the like.

In another specific embodiment, said second therapy comprises animmunomodulatory compound, wherein the immunomodulatory compound is3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione;3-(4′aminoisoindolin-1′-one)-1-piperidine-2,6-dione;4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; orα-(3-aminophthalimido) glutarimide. In a more specific embodiment, saidimmunomodulatory compound is a compound having the structure

wherein one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl, or a pharmaceutically acceptable salt,hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, ormixture of stereoisomers thereof. In another more specific embodiment,said immunomodulatory compound is a compound having the structure

wherein one of X and Y is C═O and the other is CH₂ or C═O;

R¹ is H, (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂—O₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴,(C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³,C(S)NHR³, C(O)NR³R³, C(S)NR³R³ or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl;

R³ and R^(3′) are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl,(C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, or(C₀-C₄)alkyl-(C₂-C₅)heteroaryl;

R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, or(C₂—O₅)heteroaryl;

each occurrence of R⁶ is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₂—O₅)heteroaryl, or(C₀-C₈)alkyl-C(O)O—R⁵ or the R⁶ groups can join to form aheterocycloalkyl group;

n is 0 or 1; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.In another more specific embodiment, said immunomodulatory compound is acompound having the structure

wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R is H or CH₂OCOR′;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹, R²,R³, or R⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbons

R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R′ is R⁷—CHR¹⁰—N(R⁸R⁹);

R⁷ is m-phenylene or p-phenylene or —(C_(n)—H_(2n))— in which n has avalue of 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂X₁CH₂CH₂— in which X₁ is —O—,—S—, or —NH—;

R¹⁰ (is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

Any combination of the above therapeutic agents, suitable for treatmentof inflammatory bowel disease or a symptom of inflammatory boweldisease, can be administered. Such therapeutic agents can beadministered in any combination with the placental stem cells orumbilical cord stem cells, at the same time or as a separate course oftreatment.

Placental stem cells or umbilical cord stem cells can be administered tothe individual suffering IBD, e.g., Crohn's disease, in the form of apharmaceutical composition, e.g., a pharmaceutical composition suitablefor intravenous, intramuscular or intraperitoneal injection. Placentalstem cells can be administered in a single dose, or in multiple doses.Where placental stem cells are administered in multiple doses, the dosescan be part of a therapeutic regimen designed to relieve one or moreacute symptoms of IBD, e.g., Crohn's disease, of can be part of along-term therapeutic regimen designed to prevent, or lessen theseverity, of a chronic course of the disease. In embodiments in whichplacental stem cells are administered with a second therapeutic agent,or with a second type of stem cell, the placental stem cells and secondtherapeutic agent and/or second type of stem cell can be administered atthe same time or different times, e.g., the administrations can takeplace within 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 20, 30, 40, or 50 minutes ofeach other, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or 22hours of each other, or within 1, 2, 3, 4, 5, 6, 7 8, 9 or 10 days ofeach other.

6. EXAMPLES 6.1 Example 1: Isolation of Adherent Placental Stem Cells

This example demonstrated the collection and isolation of adherentplacental stem cells.

Materials and Methods. Placenta donors were recruited from expectantmothers that enrolled in private umbilical cord blood banking programsand provided informed consent permitting the use of the exsanguinatedplacenta following recovery of cord blood for research purposes. Thesedonors permitted use of blinded data generated from the normalprocessing of their umbilical cord blood specimens for cryopreservation.This allowed comparison between the composition of the collected cordblood and the effluent perfusate recovered using this experimentalmethod described below.

Following exsanguination of the umbilical cord and placenta, theplacenta was placed in a sterile, insulated container at roomtemperature and delivered to the laboratory within 4 hours of birth.Placentas were discarded if, on inspection, they had evidence ofphysical damage such as fragmentation of the organ or avulsion ofumbilical vessels. Placentas were maintained at room temperature(23+/−2° C.) or refrigerated (4° C.) in sterile containers for 2 to 20hours. Periodically, the placentas were immersed and washed in sterilesaline at 25+/−3° C. to remove any visible surface blood or debris. Theumbilical cod was transected approximately 5 cm from its insertion intothe placenta and the umbilical vessels were cannulated with Teflon orpolypropylene catheters connected to a sterile fluid path allowingbidirectional perfusion of the placenta and recovery of the effluentfluid. The system employed herein enabled all aspects of conditioning,perfusion and effluent collection to be performed under controlledambient atmospheric conditions as well as real-time monitoring ofintravascular pressure and flow rates, core and perfusate temperaturesand recovered effluent volumes. A range of conditioning protocols wasevaluated over a 24 hour postpartum period and the cellular compositionof the effluent fluid was analyzed by flow cytometry, light microscopyand colony forming unit assays.

Placental Conditioning. A placenta was maintained under varyingconditions in an attempt to simulate and sustain a physiologicallycompatible environment for the proliferation and recruitment ofplacental stem cells. A cannula was flushed with IMDM serum-free medium(GibcoBRL, NY) containing 2 U/ml heparin (EJkins-Sinn, N.J.). Perfusionof the placenta was performed at a rate of 50 mL per minute untilapproximately 150 mL of perfusate was collected. This volume ofperfusate was labeled the “early fraction”. The placenta was perfused atthe same rate to collect a second fraction of approximately 150 mL,which was labeled the “late fraction”. During the course of theprocedure, the placenta was gently massaged to aid in the perfusionprocess and assist in the recovery of cellular material. Effluent fluidwas collected from the perfusion circuit by both gravity drainage andaspiration through the arterial cannula.

Placentas were obtained from delivery rooms along with cord blood afterobtaining written parental consent, and were processed at roomtemperature within 12 to 24 hours after delivery. Before processing, themembranes were removed and the maternal site washed clean of residualblood. The umbilical vessels were cannulated with catheters made from 20gauge Butterfly needles use for blood sample collection. Placentas werethen perfused with heparinized (2 U/mL) Dulbecco's modified Eagle Medium(HDMEM) at the rate of 15 mL/minute for 10 minutes and the perfusateswere collected from the maternal sites within one hour and the nucleatedcells counted. The perfusion and collection procedures were repeatedonce or twice until the number of recovered nucleated cells fell below100/μL. The perfusates were pooled and subjected to light centrifugationto remove platelets, debris and de-nucleated cell membranes. Thenucleated cells were then isolated by Ficoll-Hypaque density gradientcentrifugation and after washing, resuspended in HDMEM. For isolation ofadherent cells, aliquots of 5-10×10⁶ cells were placed in each ofseveral T-75 flasks and cultured with commercially available MesenchymalStem Cell Growth Medium (MSCGM) obtained from BioWhittaker, and placedin a tissue culture incubator at 37° C., 5% CO₂. After 10 to 15 days,the non-adherent cells were removed by washing with PBS, which was thenreplaced by MSCGM. The flasks were examined daily for the presence ofvarious adherent cell types and in particular, for identification andexpansion of clusters of fibroblastoid cells.

Cell Recovery and Isolation. Cells were recovered from the perfusates bycentrifugation at about 200×g for 15 minutes at room temperature. Thisprocedure served to separate cells from contaminating debris andplatelets. The cell pellets were resuspended in IMDM serum-free mediumcontaining 2 U/ml heparin and 2 mM EDTA (GibcoBRL, NY). The totalmononuclear cell fraction was isolated using Lymphoprep (Nycomed Pharma,Oslo, Norway) according to the manufacturer's recommended procedure andthe mononuclear cell fraction was resuspended. Cells were counted usinga hemocytometer. Viability was evaluated by trypan blue exclusion.Isolation of mesenchymal cells was achieved by differentialtrypsinization using a solution of 0.05% trypsin with 0.2% EDTA (Sigma).Differential trypsinization was possible because fibroblastoid cellsdetached from plastic surfaces within about five minutes whereas theother adherent populations required more than 20-30 minutes incubation.The detached fibroblastoid cells were harvested following trypsinizationand trypsin neutralization using Trypsin Neutralyzing Solution (TNS,BioWhitaker). The cells were washed in HDMEM and resuspended in MSCGM.Flow cytometry of the cells was carried out using a Becton-DickinsonFACSCalibur instrument using FITC and PE labeled monoclonal antibodiesselected on the basis of known markers for bone marrow-derived MSC(mesenchymal stem cells). Antibodies were purchased from B.D. and Caltaglaboratories (South San Francisco, Calif.), and SH2, SH3 and SH4antibody producing hybridomas were obtained from AM. Cul. andreactivities of the antibodies in their cultured supernatants weredetected by FITC or PE labeled F(ab)′₂ goat anti-mouse antibodies.Lineage differentiation was carried out using the commercially availableinduction and maintenance culture media (BioWhittaker), used as permanufacturer's instructions.

Isolation of Placental Stem Cells. Microscopic examination of theadherent cells in the culture flasks revealed morphologically differentcell types, including spindle-shaped cells, round cells with largenuclei and numerous perinuclear small vacuoles, and star-shaped cellswith several projections, through one of which the cells were attachedto the flask. No attempts were made to further characterize these typesof adherent cells, because similar non-stem cells were observed in theculture of bone marrow, cord and peripheral blood. However,fibroblastoid cells, appearing last as clusters and appearing by visualinspection to be similar to bone marrow-derived mesenchymal stem cells,were isolated by differential trypsinization and subcultured insecondary flasks. Phase microscopy of the cells, which appeared roundedafter trypsinization, showed them to be highly granulated, and similarto bone marrow-derived MSC produced in the laboratory or purchased fromBioWhittaker.

When subcultured, these adherent placental cells, in contrast to theirearlier phase, adhered within hours, assumed characteristicfibroblastoid shape, and formed a growth pattern similar to thereference bone marrow-derived MSC. Moreover, during subculturing andrefeeding, the loosely bound mononuclear cells were washed out and thecultures remained homogeneous and devoid of any visiblenon-fibroblastoid cell contaminants.

In subsequent experiments, the cell surface marker phenotype, or, in thecase of OCT-4, the gene expression phenotype, of these adherent cells,obtained from different perfusion experiments, was characterized. Theresults of these experiments are shown in Table 3, below:

TABLE 3 Characterization of placental stem cells (PLSC) collected fromseparate perfusion experiments. Frozen ID # Medium (Vials) CD34 CD45CD10 CD29 CD54 SH2 SH3 SH4 SSEA4 CD44 HLA1 CD90 Oct4 PLSC-1 BW Y (2) −− + + + + + + PLSC-2 BW Y (6) − − + + + + + + PLSC-3 BW Y (2) −− + + + + + + + PLSC-4 BW None PLSC-5 BW Y (9) − − + + + + + + PLSC-6 BWY (26) − − +/low + + + + + + PLSC-7 BW Y (2) − − + + + + + + + PLSC-8 BWY (10) − − + + + + + + + PLSC-9 BW Y (11) − − + + + + + + + PLSC-10 BW Y(10) − − + + + + + + + PLSC-11 D-5% FCS Y (9) PLSC-12 D-5% FCS Y (7)PLSC-13 D-5% FCS Y (5) PLSC-14 D-5% FCS Y (9) PLSC-15 Anthro-1 Y (7) −− + + + + + + + + + PLSC-16 Anthro-1 Y (8) − − + + + + + + + + + PLSC-17Anthro-1 Y (8) − − + + + + + + + + + PLSC-18 Anthro-1 Y (8) −− + + + + + + + + + PLSC-19 BWtoA Y (17) − − + + + + + + + + + PLSC-20BWtoA Y (40) − − + + + + + + + + + PLSC-21 BWtoA Y (9) − − + + + + + ++/− + + + PLSC-22 BWtoA FTE PLSC-23 Anthro-1 Y (10) −− + + + + + + + + + PLSC-24 Anthro-1 FTE PLSC-25 Anthro-1 FTE PLSC-26Anthro-1 Y (15) − − + + + + + + + + + PLSC-27 Anthro-1 Y (25) −− + + + + + + + + + +: Detected by flow cytometry, or, for OCT-4, geneexpression detected by RT-PCR −: Not detected Blank: Presence of markerwas not tested FTE: Failed to expand D-5% FCS: DMEM-5% FCS BWtoA: BW toAnthro-1 medium

6.2 Example 2: Culture of Placental Stem Cells

Placental stem cells are obtained from a post-partum mammalian placentaeither by perfusion or by physical disruption, e.g., enzymaticdigestion. The cells are cultured in a culture medium comprising 60%DMEM-LG (Gibco), 40% MCDB-201 (Sigma), 2% fetal calf serum (FCS)(Hyclone Laboratories), 1× insulin-transferrin-selenium (ITS), 1×lenolenic-acid-bovine-serum-albumin (LA-BSA), 10⁻⁹M dexamethasone(Sigma), 10⁻⁴M ascorbic acid 2-phosphate (Sigma), epidermal growthfactor (EGF) 10 ng/ml (R&D Systems), platelet derived-growth factor(PDGF-BB) 10 ng/ml (R&D Systems), and 100 U penicillin/1000 Ustreptomycin.

The culture flask in which the cells are cultured is prepared asfollows. T75 flasks are coated with fibronectin (FN), by adding 5 ml PBScontaining 5 ng/ml human FN (Sigma F0895) to the flask. The flasks withFN solution are left at 37° C. for 30 min. The FN solution is thenremoved prior to cell culture. There is no need to dry the flasksfollowing treatment. Alternatively, the flasks are left in contact withthe FN solution at 4° C. overnight or longer; prior to culture, theflasks are warmed and the FN solution is removed.

Placental Stem Cells Isolated by Perfusion

Cultures of placental stem cells from placental perfusate areestablished as follows. Cells from a Ficoll gradient are seeded inFN-coated T75 flasks, prepared as above, at 50-100×10⁶ cells/flask in 15ml culture medium. Typically, 5 to 10 flasks are seeded. The flasks areincubated at 37° C. for 12-18 hrs to allow the attachment of adherentcells. 10 ml of warm PBS is added to each flask to remove cells insuspension, and mixed gently. 15 mL of the medium is then removed andreplaced with 15 ml fresh culture medium. All medium is changed 3-4 daysafter the start of culture. Subsequent culture medium changes areperformed, during which 50% or 7.5 ml of the medium is removed.

Starting at about day 12, the culture is checked under a microscope toexamine the growth of the adherent cell colonies. When cell culturesbecome approximately 80% confluent, typically between day 13 to day 18after the start of culture, adherent cells are harvested by trypsindigestion. Cells harvested from these primary cultures are designatedpassage 0 (zero).

Placental Stem Cells Isolated by Physical Disruption and EnzymaticDigestion

Placental stem cell cultures are established from digested placentaltissue as follows. The perfused placenta is placed on a sterile papersheet with the maternal side up. Approximately 0.5 cm of the surfacelayer on maternal side of placenta is scraped off with a blade, and theblade is used to remove a placental tissue block measuring approximately1×2×1 cm. This placenta tissue is then minced into approximately 1 mm³pieces. These pieces are collected into a 50 ml Falcon tube and digestedwith collagenase IA (2 mg/ml, Sigma) for 30 minutes, followed bytrypsin-EDTA (0.25%, GIBCO BRL) for 10 minutes, at 37° C. in water bath.The resulting solution is centrifuged at 400 g for 10 minutes at roomtemperature, and the digestion solution is removed. The pellet isresuspended to approximately 10 volumes with PBS (for example, a 5 mlpellet is resuspended with 45 ml PBS), and the tubes are centrifuged at400 g for 10 minutes at room temperature. The tissue/cell pellet isresuspended in 130 mL culture medium, and the cells are seeded at 13 mlper fibronectin-coated T-75 flask. Cells are incubated at 37° C. with ahumidified atmosphere with 5% CO₂. Placental Stem Cells are optionallycryopreserved at this stage.

Subculturing and Expansion of Placental Stem Cells

Cryopreserved cells can be quickly thawed in a 37° C. water bath.Placental stem cells are immediately removed from the cryovial with 10ml warm medium and transferred to a 15 ml sterile tube. The cells arecentrifuged at 400 g for 10 minutes at room temperature. The cells aregently resuspended in 10 ml of warm culture medium by pipetting, andviable cell counts are determined by Trypan blue exclusion. Cells arethen seeded at about 6000-7000 cells per cm² onto FN-coated flasks,prepared as above (approximately 5×10⁵ cells per T-75 flask). The cellsare incubated at 37° C., 5% CO₂ and 90% humidity. When the cells reached75-85% confluency, all of the spent media is aseptically removed fromthe flasks and discarded. 3 ml of 0.25% trypsin/EDTA (w/v) solution isadded to cover the cell layer, and the cells are incubated at 37° C., 5%CO₂ and 90% humidity for 5 minutes. The flask is tapped once or twice toexpedite cell detachment. Once >95% of the cells are rounded anddetached, 7 ml of warm culture medium is added to each T-75 flask, andthe solution is dispersed by pipetting over the cell layer surfaceseveral times.

After counting the cells and determining viability as above, the cellsare centrifuged at 1000 RPM for 5 minutes at room temperature. Cells arepassaged by gently resuspending the cell pellet from one T-75 flask withculture medium, and evenly plating the cells onto two FN-coated T-75flasks.

Using the above methods, populations of adherent placental stem cellsare identified that express markers CD105, CD117, CD33, CD73, CD29,CD44, CD10, CD90 and CD133. This population of cells did not expressCD34 or CD45. Some, but not all cultures of these placental stem cellsexpress HLA-ABC and/or HLA-DR.

6.3 Example 3: Isolation of Placental Stem Cells from PlacentalStructures 6.3.1 Materials & Methods 6.3.1.1 Isolation of the Phenotypeof Interest

Five distinct populations of placental cells were obtained from theplacentas of normal, full-term pregnancies. All donors provided fullwritten consent for the use of their placentas for research purposes.Five populations of cells were examined: placental cells from (1)placental perfusate (from perfusion of the placental vasculature); andenzymatic digestions of (2) amnion, (3) chorion, (4) amnion-chorionplate and (5) umbilical cord cells from enzymatic digestion. The varioustissues were cleaned in sterile PBS (Gibco-Invitrogen Corporation,Carlsbad, Calif.) and placed on separate sterile Petri dishes. Thevarious tissues were minced using a sterile surgical scalpel and placedinto 50 mL Falcon Conical tubes. The minced tissues were digested with1× Collagenase (Sigma-Aldrich, St. Louis, Mo.) for 20 minutes in a 37°C. water bath, centrifuged, and then digested with 0.25% Trypsin-EDTA(Gibco-Invitrogen Corp) for 10 minutes in a 37° C. water bath. Thevarious tissues were centrifuged after digestion and rinsed once withsterile PBS (Gibco-Invitrogen Corp). The reconstituted cells were thenfiltered twice, once with 100 μm cell strainers and once with 30 μmseparation filters, to remove any residual extracellular matrix orcellular debris.

6.3.1.2 Cellular Viability Assessment and Cell Counts

The manual trypan blue exclusion method was employed post digestion tocalculate cell counts and assess cellular viability. Cells were mixedwith Trypan Blue Dye (Sigma-Aldrich) at a ratio of 1:1, and the cellswere read on hemacytometer.

6.3.1.3 Cell Surface Marker Characterization

Cells that were HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ were selected forcharacterization. Cells having this phenotype were identified,quantified, and characterized by two of Becton-Dickinson flowcytometers, the FACSCalibur and the FACS Aria (Becton-Dickinson, SanJose, Calif., USA). The various placental cells were stained, at a ratioof about 10 μL of antibody per 1 million cells, for 30 minutes at roomtemperature on a shaker. The following anti-human antibodies were used:Fluorescein Isothiocyanate (FITC) conjugated monoclonal antibodiesagainst HLA-G (Serotec, Raleigh, N.C.), CD10 (BD ImmunocytometrySystems, San Jose, Calif.), CD44 (BD Biosciences Pharmingen, San Jose,Calif.), and CD105 (R&D Systems Inc., Minneapolis, Minn.); Phycoerythrin(PE) conjugated monoclonal antibodies against CD44, CD200, CD117, andCD13 (BD Biosciences Pharmingen); Phycoerythrin-Cy5 (PE Cy5) conjugatedStreptavidin and monoclonal antibodies against CD117 (BD BiosciencesPharmingen); Phycoerythrin-Cy7 (PE Cy7) conjugated monoclonal antibodiesagainst CD33 and CD10 (BD Biosciences); Allophycocyanin (APC) conjugatedstreptavidin and monoclonal antibodies against CD38 (BD BiosciencesPharmingen); and Biotinylated CD90 (BD Biosciences Pharmingen). Afterincubation, the cells were rinsed once to remove unbound antibodies andwere fixed overnight with 4% paraformaldehyde (USB, Cleveland, Ohio) at4° C. The following day, the cells were rinsed twice, filtered through a30 μm separation filter, and were run on the flow cytometer(s).

Samples that were stained with anti-mouse IgG antibodies (BD BiosciencesPharmingen) were used as negative controls and were used to adjust thePhoto Multiplier Tubes (PMTs). Samples that were single stained withanti-human antibodies were used as positive controls and were used toadjust spectral overlaps/compensations.

6.3.1.4 Cell Sorting and Culture

One set of placental cells (from perfusate, amnion, or chorion) wasstained with 7-Amino-Actinomycin D (7AAD; BD Biosciences Pharmingen) andmonoclonal antibodies specific for the phenotype of interest. The cellswere stained at a ratio of 10 μL of antibody per 1 million cells, andwere incubated for 30 minutes at room temperature on a shaker. Thesecells were then positively sorted for live cells expressing thephenotype of interest on the BD FACS Aria and plated into culture.Sorted (population of interest) and “All” (non-sorted) placental cellpopulations were plated for comparisons. The cells were plated onto afibronectin (Sigma-Aldrich) coated 96 well plate at the cell densitieslisted in Table 4 (cells/cm²). The cell density, and whether the celltype was plated in duplicate or triplicate, was determined and governedby the number of cells expressing the phenotype of interest.

TABLE 4 Cell plating densities 96 Well Plate Culture Density of PlatedCells Conditions Sorted All All Max. Density Cell Source A Set #1: 40.6K/cm² 40.6 K/cm² 93.8 K/cm² Set #2 40.6 K/cm² 40.6 K/cm² 93.8 K/cm² Set#3: 40.6 K/cm² 40.6 K/cm² 93.8 K/cm² Cell Source B Set #1: 6.3 K/cm² 6.3K/cm² 62.5 K/cm² Set #2 6.3 K/cm² 6.3 K/cm² 62.5 K/cm² Cell Source C Set#1: 6.3 K/cm² 6.3 K/cm² 62.5 K/cm² Set #2 6.3 K/cm² 6.3 K/cm² 62.5 K/cm²

Complete medium (60% DMEM-LG (Gibco) and 40% MCDB-201 (Sigma); 2% fetalcalf serum (Hyclone Labs.); 1× insulin-transferrin-selenium (ITS); 1×linoleic acid-bovine serum albumin (LA-BSA); 10⁻⁹M dexamethasone(Sigma); 10⁻⁴ M ascorbic acid 2-phosphate (Sigma); epidermal growthfactor 10 ng/mL (R&D Systems); and platelet-derived growth factor(PDGF-BB) 10 ng/mL (R&D Systems)) was added to each well of the 96 wellplate and the plate was placed in a 5% CO₂/37° C. incubator. On day 7,100 μL of complete medium was added to each of the wells. The 96 wellplate was monitored for about two weeks and a final assessment of theculture was completed on day 12.

6.3.1.5 Data Analysis

FACSCalibur data was analyzed in FlowJo (Tree star, Inc) using standardgating techniques. The BD FACS Aria data was analyzed using the FACSDivasoftware (Becton-Dickinson). The FACS Aria data was analyzed usingdoublet discrimination gating to minimize doublets, as well as, standardgating techniques. All results were compiled in Microsoft Excel and allvalues, herein, are represented as average±standard deviation (number,standard error of mean).

6.3.2 Results 6.3.2.1 Cellular Viability

Post-digestion viability was assessed using the manual trypan blueexclusion method (FIG. 1 ). The average viability of cells obtained fromthe majority of the digested tissue (from amnion, chorion oramnion-chorion plate) was around 70%. Cells from amnion had an averageviability of 74.35%±10.31% (n=6, SEM=4.21), chorion had an averageviability of 78.18%±12.65% (n=4, SEM=6.32), amnion-chorion plate had anaverage viability of 69.05%±10.80% (n=4, SEM=5.40), and umbilical cordhad an average viability of 63.30%±20.13% (n=4, SEM=10.06). Cells fromperfusion, which did not undergo digestion, retained the highest averageviability, 89.98±6.39% (n=5, SEM=2.86).

6.3.2.2 Cell Quantification

The five distinct populations of placenta derived cells were analyzed todetermine the numbers of HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells. From theanalysis of the BD FACSCalibur data, it was observed that the amnion,perfusate, and chorion contained the greatest total number of thesecells, 30.72±21.80 cells (n=4, SEM=10.90), 26.92±22.56 cells (n=3,SEM=13.02), and 18.39±6.44 cells (n=2, SEM=4.55) respectively (data notshown). The amnion-chorion plate and umbilical cord contained the leasttotal number of cells expressing the phenotype of interest, 4.72±4.16cells (n=3, SEM=2.40) and 3.94±2.58 cells (n=3, SEM=1.49) respectively(data not shown).

Similarly, when the percent of total cells expressing the phenotype ofinterest was analyzed, it was observed that amnion and placentalperfusate contained the highest percentages of cells expressing thisphenotype (0.0319%±0.0202% (n=4, SEM=0.0101) and 0.0269%±0.0226% (n=3,SEM=0.0130) respectively (FIG. 2 ). Although umbilical cord contained asmall number of cells expressing the phenotype of interest (FIG. 2 ), itcontained the third highest percentage of cells expressing the phenotypeof interest, 0.020±0.0226% (n=3, SEM=0.0131) (FIG. 2 ). The chorion andamnion-chorion plate contained the lowest percentages of cellsexpressing the phenotype of interest, 0.0184±0.0064% (n=2, SEM=0.0046)and 0.0177±0.0173% (n=3, SEM=0.010) respectively (FIG. 2 ).

Consistent with the results of the BD FACSCalibur analysis, the BD FACSAria data also identified amnion, perfusate, and chorion as providinghigher numbers of HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells than the remainingsources. The average total number of cells expressing the phenotype ofinterest among amnion, perfusate, and chorion was 126.47±55.61 cells(n=15, SEM=14.36), 81.65±34.64 cells (n=20, SEM=7.75), and 51.47±32.41cells (n=15, SEM=8.37), respectively (data not shown). Theamnion-chorion plate and umbilical cord contained the least total numberof cells expressing the phenotype of interest, 44.89±37.43 cells (n=9,SEM=12.48) and 11.00±4.03 cells (n=9, SEM=1.34) respectively (data notshown).

BD FACS Aria data revealed that the B and A cell sources contained thehighest percentages of HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells, 0.1523±0.0227%(n=15, SEM=0.0059) and 0.0929±0.0419% (n=20, SEM=0.0094) respectively(FIG. 3 ). The D cell source contained the third highest percentage ofcells expressing the phenotype of interest, 0.0632±0.0333% (n=9,SEM=0.0111) (FIG. 3 ). The C and E cell sources contained the lowestpercentages of cells expressing the phenotype of interest,0.0623±0.0249% (n=15, SEM=0.0064) and 0.0457±0.0055% (n=9, SEM=0.0018)respectively (FIG. 3 ).

After HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells were identified and quantifiedfrom each cell source, its cells were further analyzed and characterizedfor their expression of cell surface markers HLA-G, CD10, CD13, CD33,CD38, CD44, CD90, CD105, CD117, CD200, and CD105.

6.3.2.3 Placental Perfusate-Derived Cells

Perfusate-derived cells were consistently positive for HLA-G, CD33,CD117, CD10, CD44, CD200, CD90, CD38, CD105, and CD13 (FIG. 4 ). Theaverage expression of each marker for perfusate-derived cells was thefollowing: 37.15%±38.55% (n=4, SEM=19.28) of the cells expressed HLA-G;36.37%±21.98% (n=7, SEM=8.31) of the cells expressed CD33; 39.39%±39.91%(n=4, SEM=19.96) of the cells expressed CD117; 54.97%±33.08% (n=4,SEM=16.54) of the cells expressed CD10; 36.79%±11.42% (n=4, SEM=5.71) ofthe cells expressed CD44; 41.83%±19.42% (n=3, SEM=11.21) of the cellsexpressed CD200; 74.25%±26.74% (n=3, SEM=15.44) of the cells expressedCD90; 35.10%±23.10% (n=3, SEM=13.34) of the cells expressed CD38;22.87%±6.87% (n=3, SEM=3.97) of the cells expressed CD105; and25.49%±9.84% (n=3, SEM=5.68) of the cells expressed CD13.

6.3.2.4 Amnion-Derived Cells

Amnion-derived cells were consistently positive for HLA-G, CD33, CD117,CD10, CD44, CD200, CD90, CD38, CD105, and CD13 (FIG. 5 ). The averageexpression of each marker for amnion-derived was the following:57.27%±41.11% (n=3, SEM=23.73) of the cells expressed HLA-G;16.23%±15.81% (n=6, SEM=6.46) of the cells expressed CD33; 62.32%±37.89%(n=3, SEM=21.87) of the cells expressed CD117; 9.71%±13.73% (n=3,SEM=7.92) of the cells expressed CD10; 27.03%±22.65% (n=3, SEM=13.08) ofthe cells expressed CD44; 6.42%±0.88% (n=2, SEM=0.62) of the cellsexpressed CD200; 57.61%±22.10% (n=2, SEM=15.63) of the cells expressedCD90; 63.76%±4.40% (n=2, SEM=3.11) of the cells expressed CD38;20.27%±5.88% (n=2, SEM=4.16) of the cells expressed CD105; and54.37%±13.29% (n=2, SEM=9.40) of the cells expressed CD13.

6.3.2.5 Chorion-Derived Cells

Chorion-derived cells were consistently positive for HLA-G, CD117, CD10,CD44, CD200, CD90, CD38, and CD13, while the expression of CD33, andCD105 varied (FIG. 6 ). The average expression of each marker forchorion cells was the following: 53.25%±32.87% (n=3, SEM=18.98) of thecells expressed HLA-G; 15.44%±11.17% (n=6, SEM=4.56) of the cellsexpressed CD33; 70.76%±11.87% (n=3, SEM=6.86) of the cells expressedCD117; 35.84%±25.96% (n=3, SEM=14.99) of the cells expressed CD10;28.76%±6.09% (n=3, SEM=3.52) of the cells expressed CD44; 29.20%±9.47%(n=2, SEM=6.70) of the cells expressed CD200; 54.88%±0.17% (n=2,SEM=0.12) of the cells expressed CD90; 68.63%±44.37% (n=2, SEM=31.37) ofthe cells expressed CD38; 23.81%±33.67% (n=2, SEM=23.81) of the cellsexpressed CD105; and 53.16%±62.70% (n=2, SEM=44.34) of the cellsexpressed CD13.

6.3.2.6 Amnion-Chorion Plate Placental Cells

Cells from amnion-chorion plate were consistently positive for HLA-G,CD33, CD117, CD10, CD44, CD200, CD90, CD38, CD105, and CD13 (FIG. 7 ).The average expression of each marker for amnion-chorion plate-derivedcells was the following: 78.52%±13.13% (n=2, SEM=9.29) of the cellsexpressed HLA-G; 38.33%±15.74% (n=5, SEM=7.04) of the cells expressedCD33; 69.56%±26.41% (n=2, SEM=18.67) of the cells expressed CD117;42.44%±53.12% (n=2, SEM=37.56) of the cells expressed CD10;32.47%±31.78% (n=2, SEM=22.47) of the cells expressed CD44; 5.56% (n=1)of the cells expressed CD200; 83.33% (n=1) of the cells expressed CD90;83.52% (n=1) of the cells expressed CD38; 7.25% (n=1) of the cellsexpressed CD105; and 81.16% (n=1) of the cells expressed CD13.

6.3.2.7 Umbilical Cord-Derived Cells

Umbilical cord-derived cells were consistently positive for HLA-G, CD33,CD90, CD38, CD105, and CD13, while the expression of CD117, CD10, CD44,and CD200 varied (FIG. 8 ). The average expression of each marker forumbilical cord-derived cells was the following: 62.50%±53.03% (n=2,SEM=37.50) of the cells expressed HLA-G; 25.67%±11.28% (n=5, SEM=5.04)of the cells expressed CD33; 44.45%±62.85% (n=2, SEM=44.45) of the cellsexpressed CD117; 8.33%±11.79% (n=2, SEM=8.33) of the cells expressedCD10; 21.43%±30.30% (n=2, SEM=21.43) of the cells expressed CD44; 0.0%(n=1) of the cells expressed CD200; 81.25% (n=1) of the cells expressedCD90; 64.29% (n=1) of the cells expressed CD38; 6.25% (n=1) of the cellsexpressed CD105; and 50.0% (n=1) of the cells expressed CD13.

A summary of all marker expression averages is shown in FIG. 9 .

6.3.2.8 BD FACS Aria Sort Report

The three distinct populations of placental cells that expressed thegreatest percentages of HLA ABC, CD45, CD34, and CD133 (cells derivedfrom perfusate, amnion and chorion) were stained with 7AAD and theantibodies for these markers. The three populations were positivelysorted for live cells expressing the phenotype of interest. The resultsof the BD FACS Aria sort are listed in Table 5.

TABLE 5 BD FACS Aria Sort Report Events Sorted (Phenotype of Cell SourceEvents Processed Interest) % Of Total Perfusate 135540110 51215 0.037786Amnion 7385933 4019 0.054414 Chorion 108498122 4016 0.003701

The three distinct populations of positively sorted cells (“sorted”) andtheir corresponding non-sorted cells were plated and the results of theculture were assessed on day 12. Sorted perfusate-derived cells, platedat a cell density of 40,600/cm², resulted in small, round, non-adherentcells. Two out of the three sets of non-sorted perfusate-derived cells,each plated at a cell density of 40,600/cm², resulted in mostly small,round, non-adherent cells with several adherent cells located around theperiphery of well. Non-sorted perfusate-derived cells, plated at a celldensity of 93,800/cm², resulted in mostly small, round, non-adherentcells with several adherent cells located around the well peripheries.

Sorted amnion-derived cells, plated at a cell density of 6,300/cm²,resulted in small, round, non-adherent cells. Non-sorted amnion-derivedcells, plated at a cell density of 6,300/cm², resulted in small, round,non-adherent cells. Non-sorted amnion-derived cells plated at a celldensity of 62,500/cm² resulted in small, round, non-adherent cells.

Sorted chorion-derived cells, plated at a cell density of 6,300/cm²,resulted in small, round, non-adherent cells. Non-sorted chorion-derivedcells, plated at a cell density of 6,300/cm², resulted in small, round,non-adherent cells. Non-sorted chorion-derived cells plated at a celldensity of 62,500/cm², resulted in small, round, non-adherent cells.These cells became adherent upon subsequent culture.

Subsequent to the performance of the experiments related above, andfurther culture of the placental stem cells, it was determined that thelabeling of the antibodies for CD117 and CD133, in which astreptavidin-conjugated antibody was labeled with biotin-conjugatedphycoerythrin (PE), produced background significant enough to resemble apositive reading. This background had initially resulted in theplacental stem cells being deemed to be positive for both markers. Whena different label, APC or PerCP was used, the background was reduced,and the placental stem cells were correctly determined to be negativefor both CD117 and CD133.

6.4 Example 4: Differentiation of Placental Stem Cells

Adherent placental stem cells were differentiated into several differentcell lineages. Adherent placental stem cells were isolated from theplacenta by physical disruption of tissue from anatomical sites withinthe placenta, including the amniotic membrane, chorion, placentalcotyledons, or any combination thereof, and umbilical cord stem cellswere obtained by physical disruption of umbilical cord tissue.

Placental stem cells and umbilical cord stem cells were established in amedium containing low concentrations of fetal calf serum and limitedgrowth factors. Flow cytometry analysis showed that placental stem cellstypically exhibited a CD200⁺ CD105⁺ CD73⁺ CD34⁻ CD45⁻ phenotype atpercentages of ≥70%. Placental stem cells were found to differentiatedown the adipocyte, chondrocyte and osteocyte lineages.

In an induction medium containing IBMX, insulin, dexamethasone andindomethacin, placental stem cells turned into fat laden adipocytes in 3to 5 weeks. Under osteogenic induction culture conditions, placentalstem cells were found to form bone nodules and have calcium depositionsin their extracellular matrix. Chondrogenic differentiation of placentalstem cells was performed in micropellets and was confirmed by formationof glycosaminoglycan in the tissue aggregates.

6.5 Example 5: Immunomodulation Using Placental Stem Cells

Placental stem cells possess an immunomodulatory effect, includingsuppression of the proliferation of T cells and natural killer cells.The following experiments demonstrate that placental stem cells have theability to modulate the response of T cells to stimulation in twoassays, the mixed lymphocyte reaction assay and the regression assay.

6.5.1 Mixed Lymphocyte Reaction Assays

The MLR measures the reaction of an effector population against a targetpopulation. The effectors can be lymphocytes or purified subpopulations,such as CD8⁺ T cells or NK cells. The target population is eitherallogeneic irradiated PBMCs, or as in the present studies, mature DCs.The responder population consists of allo-specific cells, estimated at20% of total T cells. The modified placental stem cell MLR usesplacental stem cells in the reaction.

Placental stem cells were plated in 96 well plate wells, and theeffector population was added. Placental stem cells and5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) stainedeffectors were preincubated for 24 hours before targets, mature DCs,were added. After six days, supernatants and non-adherent cells wereharvested. Supernatants were analyzed by Luminex bead analysis, and thecells were analyzed by flow cytometry.

Classically, the MLR produces a proliferative response in both the CD8⁺and the CD4⁺ T cell compartment. This response is a naïve T cellresponse, as two allogeneic donors have never encountered each otherbefore. Both CD4⁺ T cells and CD8⁺ T cells proliferated vigorously inthe standard MLR. When placental stem cells were added to the MLR, theCD4 and CD8 T cell proliferation, as measured by the percentage ofCFSE^(Low) responder cells, was dampened.

The effect of adding placental stem cells to an MLR (PMLR) can be seenin FIGS. 10A and 10B (PMLR trace) and FIG. 11 . The results were similarwhether only CD4⁺ or CD8⁺ T cells were used individually, or whetherequal amounts of CD4⁺ T cells and CD8⁺ T cells were used together.Placental stem cells obtained from the amnion-chorion or umbilical cordstroma suppressed the MLR to similar extents, and no difference insuppression was seen between CD4⁺ T cells and CD8⁺ T cells. This wasalso true for the bulk T cell reactions.

A separate MLR was performed using CD4⁺ T cells, CD8⁺ T cells, or bothCD4⁺ and CD8⁺ T cells, and allogeneic dendritic cells (DC). Placentalstem cells were added to the MLR, and the degree of proliferation of theT cells was assessed, using an MLR without placental stem cells as acontrol.

CD4⁺ and CD8⁺ T cells, and CD14⁺ monocytes, were isolated from buffycoats using Miltenyi MACS columns and beads, used according tomanufacturer instructions. Dendritic cells were obtained by a six-dayculture of monocytes in RPMI 1640 supplemented with 1% donor plasma,IL-4, and GM-CSF, and a two-day culture in RPMI 1640 supplemented withIL-1β, TNF-α, and IL-6. Allogeneic T cells and DC in the ratio T:DC of10:1 were incubated to produce a classic 6 day MLR. T cell proliferationwas assessed by staining T cells with CFSE (Carboxy-fluoresceindiacetate, succinimidyl ester) before being added to the assay. CSFE isused to assess the degree of proliferation by measurement of dilution ofthe stain among daughter cell populations.

To this assay, placental stem cells (PSCs) were added at the ratioT:DC:PSC of 10:1:2. The reaction was set up in a 96-well plate in afinal volume of 200 μL RPMI 1640 supplemented with 5% pooled human serum(R5). After six days, non-adherent cells were briefly resuspended andtransferred to a 5 mL tube washed with RPMI, and stained with CD4 andCD8 antibody. Proliferation of the CD4 and CD8 compartment was assessedon a BD FACS Calibur.

Placental stem cells. Placental stem cells were obtained as described inExamples 1 and 2, above. Placental stem cells were obtained from thefollowing placental tissues: amnion (AM), or amnion/chorion (AC).Umbilical cord stem cells were obtained from digestion of umbilical cord(UC). Fibroblasts (FB) and bone marrow-derived mesenchymal stem cells(MSCs) were added as controls.

Results. When placental stem cells are added to the MLR, T cellproliferation is dampened (FIG. 12 ). Placental stem cells used in theexperiments reflected in FIG. 10 were derived from one placenta,designated 61665. For all placental stem cells tested, when either CD4⁺and CD8⁺ T cells but not both were used, the CD4⁺ compartment wassuppressed to a greater degree than the CD8⁺ compartment (FIG. 12A).Suppression by AM and UC placental stem cells of CD4⁺ activation wasroughly equivalent to suppression mediated by MSCs, with a suppressionof about 60%-75%. When the MLR was run using both CD4⁺ and CD8⁺ T cells,placental stem cells suppressed proliferation in the CD4⁺ compartment toa far greater degree than the CD8⁺ compartment (FIG. 12B). Inparticular, CD4⁺ T cell proliferation suppression by AM placental stemcells approached 90%, exceeding the suppression shown by MSCs. Thedifference in suppression between these two compartments was moststriking for AM and AC placental stem cells.

Placental stem cells from different donors suppress T cell proliferationin the MLR to a different degree (FIG. 13 ). Placental stem cells from adifferent placenta, designated 65450, suppressed CD4⁺ and CD8⁺ T cellproliferation in the MLR differently than placental stem cells fromplacenta 61665. Strikingly, AC and UC PSCs from placenta 65450suppressed T cell proliferation from 80% to 95%, exceeding thesuppression in this assay by MSCs. AC placental stem cells from placenta65450, however, did not suppress T cell proliferation to an appreciabledegree (compare AM placental stem cell suppression in FIG. 10A).

Placental stem cells also suppressed the activity of Natural Killer (NK)cells in the MLR.

6.5.2 Regression Assay

Placental stem cells were shown in a regression assay to suppress a Tcell response to a B cell line expressing Epstein-Barr virus (EBV)antigens. The regression assay is a recall assay that measures effectorT cell mechanisms brought about by presentation of EBV antigen peptideson MHC Class I and II of EBV-transformed B cells. The assay is performedby mixing T cells with an artificially created transformed B cell line,the lymphoblastoid cell line (LCL) from the same donor. The LCLexpresses nine Epstein-Barr virus antigens that elicit between them arange of adaptive T and B cell responses, although in the classicregression assay, only T cell effector mechanisms are measured. Theregression assay offers a convenient way of measuring cytotoxicity totargets infected with a naturally occurring pathogen, in that the LCLexpresses the activated B cell marker CD23. Therefore, the cell count ofCD23-expressing cells is a measure of the number of LCL surviving in theassay.

The classic seventeen day regression assay gave results similar to thoseseen in the first cluster of bars in FIG. 14 . No CD23⁺ cells weredetected, as they had all been killed by CD4⁺ and CD8⁺ T cells. With theaddition of placental stem cells, seen in the next two clusters of bars,survival of CD23⁺ cells was enhanced. Without wishing to be bound bytheory, two explanations can be given for the observed effect. Eitherthe T cells had died, and left behind the LCL to expand freely, orplacental stem cells mainly increased the longevity of the LCL, havinghad less of an effect on the T cells.

In a separate regression assay, T cells and dendritic cells wereobtained from laboratory donors. Epstein-Barr virus-transformed B cellslines, LCLs, were obtained by incubating peripheral blood mononuclearcells (PBMCs) with supernatant from a lytic EBV line, B95.8, andcyclosporin A for two weeks. The LCL expressed 9 EBV antigens. Theoutgrowing LCL line is maintained in RPMI 1640 supplemented with 10%fetal calf serum. The regression assay was performed by mixing CD4⁺ orCD8⁺ T cells with autologous LCL at a ratio T:LCL of 10:1. The assay wasperformed in a 96-well plate in 200 μL RPMI 1640 supplemented with 5%pooled human serum (R5). To this assay, placental stem cells are addedin a ratio T:LCL:PSC of 10:1:2. The assay was run for 6, 10, or 17 days.

A six-day regression assay was performed using CSFE-labeled T cells.Placental stem cells from placenta 65450 suppressed T cell proliferationin the regression assay by about 65% to about 97%, a result thatcorresponds to the results for these PSCs in the MLR (FIG. 15 ). Again,UC and AC lines from placenta 65450 significantly suppressed T cellproliferation, while 65450 AM PSCs did not suppress proliferation.

In a separate experiment, it was determined that natural killer cellswere suppressed in the MLR and regression assays, as well. NK cells,when the MLR or regression assay was run including 50 U/ml IL-2, thesuppressive effect was about 45% (range about 40% to about 65%, SEM 5%).

Placental stem cells are not immunogenic. In no instance was more than5% background T cell proliferation observed against placental stem cellsfrom any donor or any placental anatomical site.

Requirement for cell-to-cell contact. The cytotoxic effect in theregression assay, and allo-recognition in the MLR, both depend on TCR (Tcell receptor):MHC interactions between target and effector cells. Therequirements for cell-to-cell contact in placental stem cell-mediatedsuppression was assessed using a transwell assay. In the assay, an MLRwas conducted in which the T cells and placental stem cells wereseparated by a membrane. As seen in FIG. 16 , the higher the number ofplacental stem cells used in the MLR, the higher the reduction ofsuppression, indicating that, particularly at higher densities,placental stem cells (UC) require significant contact with the T cellsto suppress T cell proliferation.

A separate assay confirmed that immunosuppression of T cells byplacental stem cells appears to at least partially involve a solublefactor. To determine whether the placental stem cells mediatedimmunosuppression is dependent on cell to cell contact, transwell assayswere performed in which placental stem cells were placed in an insert atthe bottom of which a membrane allowed passage only of soluble factors.At the floor of the well, separated from the placental stem cells, werethe MLR or T cells alone. In order to determine if an observed effectdepended on the relative dose of placental stem cells, the stem cellswere added at different relative densities to T cells and DCs. Whenumbilical cord placental stem cells were separated from the MLR, thesuppressive effect was partly abrogated. When placental stem cells wereused at densities similar to that used in FIG. 11 , the MLR suppressionwas abrogated 75% for CD4⁺ T cells, and 85% for CD8⁺ T cells (FIG. 17 ,FIG. 18 ). The suppressive effect was still at 66% when just a quarterdose of placental stem cells were used (UC OP 25), and dropped tobackground levels when 12,500 UC placental stem cells were added. Nochange in suppression with separation using an insert was observed (FIG.17 ). At 25,000 placental stem cells, despite the still vigoroussuppressive effect, the smallest relative drop in suppression onintroduction of the insert was observed (FIG. 18 ).

6.6 Example 6: Contact Dependence of Placental Stem CellImmunosuppression Differs from that of Bone Marrow-Derived MesenchymalStem Cells

In an experiment to determine the degree of contact dependency inimmunomodulation, umbilical cord stem cells showed a markedly differentrequirement for cell-to-cell contact for immunomodulation than that ofbone marrow derived stem cells. In particular, placental stem cellsdepended more upon cell-to-cell contact to effect immunomodulation,particularly at higher numbers of placental or mesenchymal stem cells.

Bone marrow-derived stem cells (BMSCs) and umbilical cord stem cells(UC) have different requirements for cell-to-cell contact, depending onthe ratio of adherent cells to T cells in a mixed leukocyte reactionassay (MLR). In a transwell experiment, in which placental stem cellswere separated from T cells and dendritic cells (DCs) in the MLR, thesuppression varied between the two types of adherent cell. FIG. 19displays results from the open well and transwell side by side. Whenapproximately 100,000 or 75,000 UC or BMSCs were used in the open wellformat, a similar suppression was observed. However, in the transwellformat, UCs suppress the MLR to a lesser degree than do BMSCs,indicating a larger contact dependency at these higher placental stemcell/T cell ratios. When lower placental cell to T cell ratios wereused, placental stem cells were more suppressive cell for cell.

From the suppression data, the degree of contact dependency wascalculated. FIG. 20 shows the contact dependency of the UC and BMSCMLRs. Bone marrow-derived cells are less contact dependent at higherBM/T cell ratios than are UCs. In other words, UC placental stem cellsand BMSCs behave differently with respect to an important mechanisticparameter, the need for cell-to-cell contact.

Regulatory T cells (Tregs) are necessary for BMSC-mediated T cellsuppression. See Aggarwal & Pittenger, “Human Mesenchymal Stem CellsModulate Allogeneic Immune Cell Responses,” Blood 105(4):1815-1822(2004). CD4⁺ CD25⁺ Tregs were depleted from healthy donor peripheralblood mononuclear cells (PBMCs), and a regression assay was performedusing autologous EBV (Epstein-Barr virus)-transformed cells. UCs wereadded to some conditions. As can be seen in FIG. 21 , there is nodifference in placental stem cell-mediated suppression of the T cellresponse in the regression assay whether or not Tregs are present. Thus,while T regulatory T cells are reportedly necessary for BMSC-mediated Tcell suppression, T regulatory cells do not appear to play a role inplacental stem cell-mediated immune suppression.

An MLR was performed in which the T cells were taken from an MLRsuppressed by placental stem cells, and the dendritic cells were addedfresh. The T cells were stained with CFSE, which is distributed equallyinto daughter cells during proliferation. CFSE^(Hi) cells are T cellsthat have not proliferated (e.g., the left-most peaks in the panels inFIG. 21 ). This population was obtained by sorting stained T cells on aFACS Aria. These cells were used in a second MLR with fresh dendriticcells. As can be seen in FIG. 22 , no lasting suppression was observed,as the formerly suppressed cells proliferated well against the DCs. Itis unlikely that the CFSE^(Lo) cells (that is, daughter cells) wouldhave been responsible for the suppression, as these cells themselvesproliferated subsequently. The CFSE^(Hi) population is made up ofnon-allo-specific cells that would not have proliferated against this DCdonor, as well as T cells suppressed by placental stem cells. Once theplacental stem cells were removed, the suppressed cells proliferated.

An MLR is suppressed by BMSCs when approximately 10% of the supernatantis replaced by the supernatant from a BMSC MLR. In sharp contrast, nochange in T cell proliferation was observed when supernatant wasreplaced by supernatant from an MLR comprising placental stem cells,even when 75% of the medium was replaced (FIG. 23 ).

It is possible that DCs or resting T cells are affected by incubationwith placental stem cells for different amounts of time before startingthe MLR. This was tested by incubating placental stem cells or BMSCswith T cells (FIG. 24A) or DCs (FIG. 24B) for varying lengths of timebefore starting the assay. Preincubating T cells and placental stemcells does not alter the suppressive phenotype appreciably (FIG. 20A).However, BMSC T cell suppression changes depending of the length ofDC/placental stem cell preincubation. As shown in FIG. 20B, suppressionby BMSCs is strongest when DCs are added one day after the T cells. Amuch lower suppression appears, however, when DCs are added at the sametime as T cells. Incubating DCs longer with BMSCs can reverse this lossof suppression. At two days preincubation, the suppression approachesthe scenario where DCs are added a day after T cells (+1 day). Nosimilar tendency is observed with placental stem cell-mediatedsuppression.

6.7 Example 7: Cytokine Profile of Placental Stem Cells and UmbilicalCord Stem Cells in the MLR and Regression Assay

Umbilical cord stem cells (UC) and placental stem cells from amnionchorion plate (AC) were determined to secrete certain cytokines into theMLR medium.

In some assays, a cytokine array was used to measure the levels ofcytokines and chemokines in the supernatants. Several factors were foundto be secreted into supernatants, the most relevant to the MLR andregression assays being macrophage inflammatory protein (MIP)-1α andMIP-1β. Both of these chemoattractants attract T cells, and are secretedby CD8⁺ T cells in response to human immunodeficiency virus (HIV)infection. When assayed in the MLR, these chemoattractants' secretioncorrelated inversely with placental stem cell and MSC suppression of theMLR (FIG. 25 ). Neither placental stem cells nor MSCs secreted MIP-1αand MIP-1β.

In another study, a correlation was found in secretion of MCP-1 andIL-6, both of which are important immuno-regulators (FIG. 26 and FIG. 27; compare with FIG. 11 ). While placental stem cells alone secreted noIL-6 or MCP-1, the UC and AC lines, both of which suppress the MLR and Tcell proliferation in the regression assay (FIG. 11 ), secrete MCP-1 andIL-6 (FIG. 26 and FIG. 27 ). Although IL-6 is mostly associated withpro-inflammatory actions (see, e.g., Kishimoto et al., Annu. Rev.Immunol. 23:1-21 (2005)), it also has other functions, such as aprotective role during liver damage in mice (see, e.g., Klein et al., J.Clin. Invest 115:860-869 (2005)).

In a separate study, AC used in an MLR or regression assay were analyzedfor cytokine secretion. Cytokines were measured on a Luminex system insupernatants from 6-day stem cell cultures, stem cell MLRs or stem cellregression assays. MLRs included the stem cells, dendritic cells (DC),and T cells in a ratio of 2/1/10. Epstein-Barr virus (EBV) regressionassays included stem cells, EBV tumor cells (Ts), and T cells at TS:stemcell:T ratio of 2:1:10.

Levels of IL-6 (11 ng/ml) and IL-8 (16 ng/ml) were found to stayconstant in stem cell solo cultures, MLRs, and regression assays. Theconcentration of MCP-1 was determined to be about 2 ng/ml in stem cellsolo cultures and non-suppressive control adherent cell MLRs andregression assays, but increased to about 10 ng/ml in suppressed stemcell MLRs and stem cell regression assays. These values fall withinserum levels recorded for MCP-1.

Interleukin-2 (IL-2) is both a T cell survival factor and an obligatefactor for CD4⁺ CD25⁺ T regulatory cells. This T cell subset is notrequired for T cell suppression by the AC stem cells, but IL-2 levelsconsistently decrease during MLR suppression by AC stem cells. MLRsupernatants in the absence of AC stem cells contained about 35 pg/mlIL-2, whereas the MLRs that included AC stem cells contained up to 440pg/ml IL-2.

The IL-2 concentrations correlated with suppression. For example, a CD4⁺T cell MLR showing 85% suppression contained 330 pg/ml IL-2, and a CD8⁺T cell MLR showing 85% suppression, using AC stem cells contained 66pg/ml IL-2. These results indicate that IL-2 and MCP-1, traditionallyknown as stimulators of the immune response, may play a role in immunesuppression.

6.8 Example 8: Production of Cryopreserved Stem Cell Product and StemCell Bank

This Example demonstrates the isolation of placental stem cell and theproduction of a frozen stem cell-based product.

Summary: Placental tissue is dissected and digested, followed by primaryand expansion cultures to achieve an expanded cell product that producesmany cell doses. Cells are stored in a two-tiered cell bank and aredistributed as a frozen cell product. All cell doses derived from asingle donor placenta are defined as a lot, and one placenta lot isprocessed at a time using sterile technique in a dedicated room andClass 100 laminar flow hood. The cell product is defined as beingCD105⁺, CD200⁺, CD10⁺, and CD34⁻, having a normal karyotype and no orsubstantially no maternal cell content.

6.8.1 Obtaining Stem Cells

Tissue Dissection and Digestion: A placenta is obtained less than 24hours after expulsion. Placental tissue is obtained from amnion, acombination of amnion and chorion, or chorion. The tissue is minced intosmall pieces, about 1 mm in size. Minced tissue is digested in 1 mg/mlCollagenase 1A for 1 hour at 37° C. followed by Trypsin-EDTA for 30minutes at 37° C. After three washes in 5% FBS in PBS, the tissue isresuspended in culture medium.

In another embodiment, a placenta is obtained les than 24 hours afterexpulsion. After cleaning the placenta, a hemostat is clamped to thedistal end of the umbilical cord. The umbilical cord is cut at thejunction with the placenta and transferred to a sterile dish. Aftercutting the cord below the hemostat, the cord is massaged to removeblood clots, and transferred to 500 ml PBS containing gentamicin andamphotericin B. 5 g of this cord is used. A scalpel is used to trim theremaining placental material by cutting in a radius of about 3 inchesfrom the umbilical cord attachment point. Blood clots are forced fromthe remaining material, and 5 g of the amnion-chorion, centered at theumbilical cord root, is transferred to the same container as theumbilical cord. The umbilical cord and amnion-chorion tissue is sliced,then minced to pieces about 1 mm³ in size. The tissue is then digestedwith 1 mg/ml Collagenase 1A (20 ml/g tissue) for 1 hour at 37° C.followed by Trypsin-EDTA (10 ml/g tissue) for 30 minutes at 37° C. Afterthree washes in 5% FBS in PBS, the tissue is resuspended in culturemedium (20 ml/g tissue) and transferred to T flasks at about 0.22ml/cm².

Primary Culture: The purpose of primary culture is to establish cellsfrom digested placental tissue. The digested tissue is suspended inculture medium and placed into Corning T-flasks, which are incubated ina humidified chamber maintained at 37° C. with 5% CO₂. Half of themedium is replenished after 5 days of culture. High-density colonies ofcells form by 2 weeks of culture. Colonies are harvested withTrypsin-EDTA, which is then quenched with 2% FBS in PBS. Cells arecentrifuged and resuspended in culture medium for seeding expansioncultures. These cells are defined as Passage 0 cells having doubled 0times.

Expansion Culture: Cells harvested from primary culture, harvested fromexpansion culture, or thawed from the cell bank are used to seedexpansion cultures. Cell Factories (NUNC™) are treated with 5% CO₂ inair at 50 ml/min/tray for 10 min through a sterile filter and warmed ina humidified incubator maintained at 37° C. with 5% CO₂. Cell seeds arecounted on a hemacytometer with trypan blue, and cell number, viability,passage number, and the cumulative number of doublings are recorded.Cells are suspended in culture medium to about 2.3×10⁴ cells/ml and 110ml/tray are seeded in the Cell Factories. After 3-4 days and again at5-6 days of culture, culture medium is removed and replaced with freshmedium, followed by another treatment with 5% CO₂ in air. When cellsreach approximately 10⁵ cells/cm², cells are harvested withTrypsin-EDTA, followed by quenching with 2% FBS in PBS. Cell are thencentrifuged and resuspended in culture medium.

Cryopreservation: Cells to be frozen down are harvested from culturewith Trypsin-EDTA (e.g., 0.044 ml/cm² for 5 minutes), quenched with 2%FBS in PBS, and counted on a hemacytometer. After centrifugation (e.g.,at 300×g), cells are resuspended with 10% DMSO in FBS to a concentrationof about 1 million cells/ml for cells to be used for assembly of a cellbank, and 10 million cells/ml for individual frozen cell doses. Inanother embodiment, the cells are diluted to about 2 million calls/ml in10% HAS, 10% DMSO in Plasmalyte. The cell solution is transferred to afreezing container, which is placed in an isopropyl alcohol bath in a−80° C. freezer. The following day, cells are transferred to liquidnitrogen.

6.8.2 Design of a Stem Cell Bank

A “lot” is defined as all cell doses derived from a single donorplacenta. Cells maintained normal growth, karyotype, and cell surfacemaker phenotype for over 8 passages and 30 doublings during expansionculture. Given this limitation, doses comprise cells from 5 passages andabout 20 doublings. To generate a supply of equivalent cells, a singlelot is expanded in culture and is stored in a two-tiered cell bank andfrozen doses. In particular, cells harvested from the primary culture,which are defined as Passage 0 cells having undergone 0 doublings, areused to initiate an expansion culture. After the first passage,approximately 4 doublings occur, and cells are frozen in a Master CellBank (MCB). Vials from the MCB are used to seed additional expansioncultures. After two additional passages of cells thawed from the MCB,cells are frozen down in a Working Cell Bank (WCB), approximately 12cumulative doublings. Vials from the WCB are used to seed an expansionculture for another 2 passages, resulting in Passage 5 cells atapproximately 20 doublings that are frozen down into individual doses.

6.8.3 Thawing Cells for Culture

Frozen containers of cells are placed into a sealed plastic bag andimmersed in a 37° C. water bath. Containers are gently swirled until allof the contents are melted except for a small piece of ice. Containersare removed from the sealed plastic bag and a 10× volume of culturemedium is slowly added to the cells with gentle mixing. A sample iscounted on the hemacytometer and seeded into expansion cultures.

6.8.4 Thawing Cells for Injection

Frozen containers of cells are transferred to the administration site ina dry nitrogen shipper. Prior to administration, containers are placedinto a sealed plastic bag and immersed in a 37° C. water bath.Containers are gently swirled until all of the contents are meltedexcept for a small piece of ice. Containers are removed from the sealedplastic bag and an equal volume of 2.5% HSA/5% Dextran is added. Cellsare injected with no further washing.

6.8.5 Testing and Specifications

A maternal blood sample accompanies all donor placentas. The sample isscreened for Hepatitis B core antibody and surface antigen, Hepatitis CVirus antibody and nucleic acid, and HIV I and II antibody and nucleicacid. Placental processing and primary culture begins prior to thereceipt of test results, but continues only for placentas associatedwith maternal blood samples testing negative for all viruses. A lot isrejected if the donor tests positive for any pathogen. In addition, thetests described in Table 6 are performed on the MCB, the WCB, and asample of the cell dose material derived from a vial of the WCB. A lotis released only when all specifications are met.

TABLE 6 Cell testing and specifications Test Methods Required ResultSterility BD BACTEC PEDS Negative PLUS/F and BACTEC Myco/F LyticEndotoxin LAL gel clot ≤5 EU/ml* Viability Trypan Blue >70% viableMycoplasma Direct culture, DNA- Negative fluorochrome (FDA PTC 1993)Identity Flow cytometry CD105⁺, CD200⁺, (see below) CD10⁺, CD34⁻ CellPurity Microsatellite No contaminating cell detected Karyotype G-bandingand Normal chromosome count on metaphase cells *For the product designedto be 40 ml of frozen cells/dose and a maximum of 5 EU/ml, the cellproduct is below the upper limit of 5 EU/kg/dose for recipients over 40kg in body weight.

6.8.6 Surface Marker Phenotype Analysis

Cells are placed in 1% paraformaldehyde (PFA) in PBS for 20 minutes andstored in a refrigerator until stained (up to a week). Cells are washedwith 2% FBS, 0.05% sodium azide in PBS (Staining Buffer) and thenresuspended in staining buffer. Cells are stained with the followingantibody conjugates: CD105-FITC, CD200-PE, CD34-PECy7, CD10-APC. Cellsare also stained with isotype controls. After 30 minute incubation, thecells are washed and resuspended with Staining Buffer, followed byanalysis on a flow cytometer. Cells having an increased fluorescencecompared to isotype controls are counted as positive for a marker.

6.9 Treatment of Immune-Related Diseases Using Placental Stem Cells orUmbilical Cord Stem Cells

This Example provides example treatment regimens for immune-relateddiseases or conditions.

6.9.1 Treatment of Crohn's Disease

An individual presents with ileocolitis, a form of Crohn's disease, andis experiencing abdominal pain, bloody diarrhea, and fever. Theindividual is administered 1-5×10⁸ CD10⁺ CD34⁻ CD105⁺ CD200⁺ placentalstem cells and/or umbilical cord stem cells in 0.9% NaCl solutionintravenously. The individual is monitored over the subsequent two weeksto assess reduction in one or more of the symptoms. The individual ismonitored over the course of the next year, and placental stem cells inthe same dose are administered as needed.

6.9.2 Treatment of Graft-Versus-Host Disease

An individual awaiting an allogeneic bone marrow transplant isadministered 5-10×10⁸ CD10⁺ CD34⁻ CD105⁺ CD200⁺ placental stem cellsand/or umbilical cord stem cells in 0.9% NaCl solution intravenouslywithin 24 hours prior to bone marrow transplantation. Administration ofthe stem cells is repeated within 24 hours after bone marrowtransplantation. The individual is monitored over the next 100 days, andis administered a follow-up dose of 5-10×10⁸ CD10⁺ CD34⁻ CD105⁺ CD200⁺placental stem cells and/or umbilical cord stem cells is GVHD developsand progresses beyond Grade I.

6.9.3 Treatment of Rheumatoid Arthritis

An individual presents with rheumatoid arthritis in three or morejoints. The individual is administered a combination of placental orumbilical cord stem cells and placental stem cells that have beenmodified to produce a fusion polypeptide comprising IL-1Ra and DHFR,wherein the two types of stem cells are administered in a 1:1 ratio. Theengineered and non-engineered cells are 1-5×10⁸ CD10⁺ CD34⁻ CD105⁺CD200⁺ placental stem cells and/or umbilical cord stem cells in 0.9%NaCl solution. The individual is given methotrexate at a standard dosageand monitored for reduction in joint inflammation.

EQUIVALENTS

The compositions and methods disclosed herein are not to be limited inscope by the specific embodiments described herein. Indeed, variousmodifications of the compositions and methods in addition to thosedescribed will become apparent to those skilled in the art from theforegoing description and accompanying figures. Such modifications areintended to fall within the scope of the appended claims.

Various publications, patents and patent applications are cited herein,the disclosures of which are incorporated by reference in theirentireties.

What is claimed:
 1. A method of treating an individual having or at riskof developing a disease, disorder or condition associated with or causedby an inappropriate or unwanted immune response, comprisingadministering to the individual a therapeutically effective amount ofplacental stem cells, or culture medium conditioned by placental stemcells, wherein the therapeutically effective amount is an amountsufficient to cause a detectable improvement in one or more symptoms ofsaid disease, disorder or condition, wherein said disease, disorder orcondition is scleroderma, and wherein said placental stem cell is aCD10+, CD34−, CD105+, CD200+ placental stem cell.
 2. The method of claim1, wherein said placental stem cells express CD200 and HLA-G, or expressCD73, CD105, and CD200, or express CD200 and OCT-4, or express CD73,CD105 and HLA-G, or express CD73 and CD105 and facilitate the formationof one or more embryoid-like bodies in a population of placental cellscomprising said stem cell when said population is cultured underconditions that allow for the formation of an embryoid-like body, orexpress OCT-4 and facilitate the formation of one or more embryoid-likebodies in a population of placental cells comprising said stem cell whensaid population is cultured under conditions that allow for theformation of an embryoid-like body.
 3. The method of claim 1, comprisingadministering a second therapeutic agent to said individual.
 4. Themethod of claim 3, wherein said second therapeutic agent is ananti-inflammatory drug, a proton pump inhibitor, an immunosuppressantcompound, or a vasodilator.
 5. The method of claim 3, wherein saidsecond therapeutic agent is an anti-inflammatory drug, animmunosuppressant compound, exposure to sunlight, exposure toultraviolet light, a topical steroid, local superficial radiotherapy,total skin electron beam radiation, application of organic honey to skinaffected by erythroderma, an interferon, a retinoid, a rexinoid, orvorinostat.